The use of deuterium oxide (D2O) has greatly expanded the scope of what is possible for the measurement of protein synthesis. The greatest asset of D2O labeling is that it facilitates the measurement of synthesis rates over prolonged periods of time from single proteins through integrated tissue-based measurements. Because the ease of administration, the method is amenable for use in a variety of models and conditions. Although the method adheres to the same rules as other isotope methods, the flexibility can create conditions that are not the same as other approaches and thus requires careful execution to maintain validity and reliability. For this CORP article, the assembled authors provide a history that gave rise to the method, discuss the advantages and disadvantages of the method, the critical assumptions, guidelines, and best practices based on instrumentation, models, and experimental design. The goal of this CORP article is to propagate additional use of D2O in a manner that produces reliable and valid data.