In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (²H₂O) labeling can safely maintain stable body water ²H enrichments for weeks or months. ²H is metabolically incorporated into C–H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so ²H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of ²H₂O-labeled rodent tissue proteins that metabolic ²H flux into C–H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By ²H₂O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl₄ was detected. Kinetics of several human serum proteins were also measured, reproducing published t_1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion, ²H₂O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.