Increasing interest in measuring, modelling and valuing ecosystem services (ES), the benefits that ecosystems provide to people, has resulted in the development of an array of ES assessment tools in recent years. Selecting an appropriate tool for measuring and modelling ES can be challenging. This document provides guidance for practitioners on existing tools that can be applied to measure or model ES provided by important sites for biodiversity and nature conservation, including Key Biodiversity Areas, natural World Heritage sites, and protected areas. This guidance builds on existing reviews of ES assessment tools but has an explicit focus on assessing ES for sites of importance for biodiversity and nature conservation. Key Biodiversity Areas (KBAs) are sites contributing significantly to the global persistence of biodiversity. Natural World Heritage sites (WHS) are natural features, formations and areas which, because of their exceptional qualities, are considered to be of ‘Outstanding Universal Value’ and therefore merit special protection. Protected areas (PAs) are clearly defined geographical spaces, recognised, dedicated and managed through legal or other effective means, to achieve the long-term conservation of nature with associated ecosystem services and cultural values. Information about ES provided by KBAs, WHS and PAs can be useful for many reasons, including increasing support for safeguarding the multiple benefits provided by sites, informing management decisions, ensuring equity in resource use and benefits sharing, and enabling evaluation of the consequences of management or policy changes on ES provided by the sites. In this guide, we summarise a range of possible reasons for ES assessment and identify tools that can be used for each purpose. We review the importance of scoping the purpose and objectives of the ES assessment in guiding tool selection and in engaging stakeholders. We also differentiate between qualitative and quantitative ES assessment and when each type of assessment (or both) might be useful. We compare a set of nine ES assessment tools that are (a) most commonly applied, (b) available to practitioners at no cost, and (c) can be applied in new contexts (i.e. they are not restricted to specific countries or case studies). These nine were selected from a broader review of 30 tools, and we provide links to resources where information on a larger number of ES assessment tools and approaches can be found. We divide the nine tools into two types: written step-by-step tools and computer-based modelling tools. Selecting an appropriate tool for ES assessment is informed by three factors: (1) purpose of the assessment, (2) required outputs (qualitative or quantitative, spatial or non-spatial, monetary or non-monetary), and (3) practical considerations such as time, budget and data availability. We provide a series of comparisons of ES tools according to each of these considerations along with decision trees that can help guide a practitioner to a tool based on assessment objectives, practical considerations, and the type of output desired. We also summarise which ecosystem services can be assessed using each of the tools and the key differences between them. In the Annexes, we provide a short description of each of the nine reviewed tools and case studies demonstrating how they have been applied to assess ES within a KBA, WHS or PA. Three of the tools reviewed—EST, TESSA, and PA-BAT—are PDF documents that walk users step-by-step through an ES assessment. The EST is a guidance document consisting of steps with practical worksheets for conducting qualitative and/ or quantitative ES assessment, indicators, advice on relevant issues, and a compendium of tools, methods, and models that might be applied. The PA-BAT is a rapid, workshop-driven and standardised assessment of different stakeholders’ perceptions about ES benefits from protected and other areas. TESSA is a PDF manual that provides accessible guidance and low-cost methods to evaluate the benefits people receive from nature at particular sites. The other six tools reviewed are computer-based modelling tools. ARIES and MIMES are modelling platforms, which can incorporate scenarios, spatial assessment and economic valuation of ES and integrate different ecological and economic models to understand and visualise ES values. InVEST is a suite of software models with defined model parameters for mapping and quantifying ES in biophysical or economic terms under different scenarios for which the user must simply provide the input data. CostingNature and WaterWorld are web-based tools for spatially analysing ES which provide model parameters and all the required input datasets and the user needs only to specify an area of interest and choose from pre-selected scenarios (e.g. land use and climate change) or design their own. SolVES is an ArcGIS-dependent application that allows the user to identify, assess and map the perceived social values that people attribute to cultural ES, which requires conducting stakeholder surveys and running models to produce spatial outputs. Selecting an appropriate tool requires identifying the specific question being addressed, what sorts of results or outputs are required, and consideration of practical factors such as the level of expertise, time and data required for applying any given tool. While each tool is different, they all provide an opportunity to shed light on ecosystem services issues and support management and policy decisions.