Water balance measurements are the simplest and most direct means of estimating evapotranspiration (ET). However, numerous factors relating to climate and terrain characteristics contribute to the variability that makes the assessment of evapotranspiration challenging at the ecosystem or even the plot scale. Alternative methods, such as an isotope mass balance (IMB), can provide evapotranspiration estimates. This paper illustrates two IMB examples of partitioning evaporation and transpiration. The first example demonstrates at the laboratory scale how accurate mass-balance measurements provide a complete validation and refinement of the isotope mass balance methods. The second IMB case uses similar data processing methods for an experimental field design. These methods are further validated by comparison with previous laboratory and field studies. Finally, this paper presents a comparison between partitioned ET ratios from a nearby U.S. Geological Survey (USGS) microclimate site produced using the Flux Variance Similarity (FVS) method. The results suggest the potential of employing these methods to estimate evaporation and transpiration source contributions at various scales. This technique and its further development show IMB methods are an appropriate tool for partitioning evapotranspiration.