There is currently no direct, facile method to determine total-body skeletal muscle mass for the diagnosis and treatment of skeletal muscle wasting conditions such as sarcopenia, cachexia, and disuse. We tested in rats the hypothesis that the enrichment of creatinine-(methyl-d₃) (D₃-creatinine) in urine after a defined oral tracer dose of D₃-creatine can be used to determine creatine pool size and skeletal muscle mass. We determined 1) an oral tracer dose of D₃-creatine that was completely bioavailable with minimal urinary spillage and sufficient enrichment in the body creatine pool for detection of D₃-creatine in muscle and D₃-creatinine in urine, and 2) the time to isotopic steady state. We used cross-sectional studies to compare total creatine pool size determined by the D₃-creatine dilution method to lean body mass determined by independent methods. The tracer dose of D₃-creatine (<1 mg/rat) was >99% bioavailable with 0.2–1.2% urinary spillage. Isotopic steady state was achieved within 24–48 h. Creatine pool size calculated from urinary D₃-creatinine enrichment at 72 h significantly increased with muscle accrual in rat growth, significantly decreased with dexamethasone-induced skeletal muscle atrophy, was correlated with lean body mass (r = 0.9590; P < 0.0001), and corresponded to predicted total muscle mass. Total-body creatine pool size and skeletal muscle mass can thus be accurately and precisely determined by an orally delivered dose of D₃-creatine followed by the measurement of D₃-creatinine enrichment in a single urine sample and is promising as a noninvasive tool for the clinical determination of skeletal muscle mass.