Endogenous creatinine clearance (Ccr) is widely accepted as an estimate of glomerular filtration rate (GFR), the best overall biomarker of kidney function. However, current common methods of measuring creatinine are not sensitive enough for mouse plasma. Accordingly, we here report a new method of measuring creatinine by liquid chromatography tandem mass spectrometry (LC-MS/MS) using deuterated [²H₃]-creatinine as an internal standard. The assay requires 10 μl or less of plasma or urine, and is eight times more sensitive than high-performance liquid chromatography. The reproducibility of the assay of replicates is approximately ±10%. The plasma creatinine levels of wild type male C57BL/6J mice obtained by LC-MS/MS are 0.076±0.002 mg/dl (n=65). To estimate daily urinary creatinine excretion for calculating Ccr, we collected urine from mice housed in metabolic cages, and combined this with washes from the cage internal surfaces. Creatinine in the wash varies from 4 to 67% of the total daily urinary creatinine excretion (typically ∼400 μg/day). Ccr obtained by LC-MS/MS was 329±17 μl/min, which is indistinguishable from GFR measured by using fluorescein isothiocyanate-inulin. The LC-MS/MS method is sensitive, specific, simple, fast, and inexpensive; it is suitable for estimating GFR in conscious mice or other small animals. As it allows repeated measurements in the same animals, it facilitates detection of subtle differences or changes in renal function.