In water-limited ecosystems, partitioning ecosystem-scale evapotranspiration fluxes between plant transpiration and soil/canopy evaporation remains a theoretical and technical challenge. We used the Biosphere 2 glasshouse to assess partitioning of evapotranspiration across an experimentally manipulated gradient of woody plant cover using continuous measurements of near-surface variations in the stable isotopic composition of water vapor (δ2H). Our technique employs a newly-developed laser-based isotope analyzer and the Keeling plot approach for surface flux partitioning. The applicability of the technique was verified by comparison to separate, simultaneous lysimeter and sap flow estimates of ET partitioning. The results showed an expected increase in fractional contribution of transpiration to evapotranspiration as woody cover increased—from T/ET = 0.61 at 25% woody cover to T/ET = 0.83 at 100% cover. Further development of this technique may enable field characterization of evapotranspiration partitioning across diverse woody cover gradients, a central issue in addressing dryland ecohydrological responses to land use and climate change.