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<p>Evaporation (<span class="inline-formula"><i>E</i></span>) and transpiration (<span class="inline-formula"><i>T</i></span>) respond differently to ongoing changes in climate, atmospheric composition, and land use. It is difficult to partition ecosystem-scale evapotranspiration (ET) measurements into <span class="inline-formula"><i>E</i></span> and <span class="inline-formula"><i>T</i></span>, which makes it difficult to validate satellite data and land surface models. Here, we review current progress in partitioning <span class="inline-formula"><i>E</i></span> and <span class="inline-formula"><i>T</i></span> and provide a prospectus for how to improve theory and observations going forward. Recent advancements in analytical techniques create new opportunities for partitioning <span class="inline-formula"><i>E</i></span> and <span class="inline-formula"><i>T</i></span> at the ecosystem scale, but their assumptions have yet to be fully tested. For example, many approaches to partition <span class="inline-formula"><i>E</i></span> and <span class="inline-formula"><i>T</i></span> rely on the notion that plant canopy conductance and ecosystem water use efficiency exhibit optimal responses to atmospheric vapor pressure deficit (<span class="inline-formula"><i>D</i></span>). We use observations from 240 eddy covariance flux towers to demonstrate that optimal ecosystem response to <span class="inline-formula"><i>D</i></span> is a reasonable assumption, in agreement with recent studies, but more analysis is necessary to determine the conditions for which this assumption holds. Another critical assumption for many partitioning approaches is that ET can be approximated as <span class="inline-formula"><i>T</i></span> during ideal transpiring conditions, which has been challenged by observational studies. We demonstrate that <span class="inline-formula"><i>T</i></span> can exceed 95&thinsp;% of ET from certain ecosystems, but other ecosystems do not appear to reach this value, which suggests that this assumption is ecosystem-dependent with implications for partitioning. It is important to further improve approaches for partitioning <span class="inline-formula"><i>E</i></span> and <span class="inline-formula"><i>T</i></span>, yet few multi-method comparisons have been undertaken to date. Advances in our understanding of carbon–water coupling at the stomatal, leaf, and canopy level open new perspectives on how to quantify <span class="inline-formula"><i>T</i></span> via its strong coupling with photosynthesis. Photosynthesis can be constrained at the ecosystem and global scales with emerging data sources including solar-induced fluorescence, carbonyl sulfide flux measurements, thermography, and more. Such comparisons would improve our mechanistic understanding of ecosystem water fluxes and provide the observations necessary to validate remote sensing algorithms and land surface models to understand the changing global water cycle.</p>