The analysis of the stable isotopic composition of hydrogen and oxygen in water samples from soils and plants can help to identify sources of vegetation water uptake. This approach requires that the heterogeneous nature of plant and soil matrices is carefully accounted for during experimental design, sample collection, water extraction and analyses. The comparability and shortcomings of the different methods for extracting water and analyzing isotopic composition have been discussed in specialized literature. Yet, despite insightful comparisons of extraction methods and benchmarking methodologies of laboratories worldwide, the community still lacks a roadmap to guide sample collection, extraction, and isotopic analyses, and many practical issues for potential users remain unresolved: for example, which (soil or plant) water pool(s) does the extracted water represent? These constitute a hurdle for the implementation of the approach by newcomers. Here, we summarize discussions led in the framework of the COST Action WATSON (“WATer isotopeS in the critical zONe: from groundwater recharge to plant transpiration”—CA19120). We provide guidelines for (1) sampling soil and plant material for isotopic analysis, (2) methods for laboratory or in situ water extraction, and (3) measurements of isotopic composition. We highlight the importance of considering the process chain as a whole, from experimental design to isotopic analysis to minimize biased estimates of the relative contribution of different water sources to plant water uptake. We conclude by acknowledging some of the limitations of this methodology and advice on the collection of key environmental parameters prior to sample collection for isotopic analyses. This article is categorized under: Science of Water > Hydrological Processes Science of Water > Water and Environmental Change Science of Water > Water Extremes.