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Objective To investigate the potentiality of D3-creatine dilution method to be used in detection of skeletal muscle mass, so as to provide a new method for skeletal muscle mass detection in mice. Methods Four weeks, 10 weeks and 13 months old male C57BL/6 mice (10 each) were used in the present study. All mice were fed with a single dose of D3-creatine (2mg/kg) after testing body composition by EchoMRITM. Urine was collected at 24h, 48h and 72h after creatine administration, and analyzed by liquid chromatography-tandem mass spectrometry mass spectrometry (LC-MS/MS) that was used to determine the creatine pool size. The relationship between creatine pool size and the skeletal muscle mass determined by EchoMRITM was analyzed. Results Both lean body mass and total creatine pool size were aged mice > adult mice > juvenile mice. There was a significant correlation (r=0.687, P=0.000) between the lean body mass measured by EchoMRITM and the total creatine pool size calculated by D3-creatine dilution method. Conclusion The D3-creatine dilution method may be used to determine the skeletal muscle mass of mice. DOI: 10.11855/j.issn.0577-7402.2017.07.10