Determination of 1-methylhistidine (1-MH) and 3-methylhistidine (3-MH) is important to monitor muscle protein catabolism. Here, an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for quantification of 1-MH and 3-MH in human urine is described.Urine samples were prepared by dilution with water after addition of isotopic internal standard. The samples were chromatographed on a SB-aq (2.1×50mm, 1.8μm) column with acetonitrile −0.1% formic acid in water (2:98, v/v) as mobile phase. Mass spectrometric detection was performed on a triple quadrupole mass spectrometer using positive electrospray ionization (ESI). 1-MH and 3-MH were monitored by the following transitions: 1-MH, m/z 170.1→ m/z 126.1; 3-MH, m/z 170.1→ m/z 124.1.For 1-MH and 3-MH, calibration curves were linear over the concentration range of 5–500nmol/ml. The lower limit of quantification was 5nmol/ml. The accuracy was within 85%–115% and precision was <15%. 1-MH and 3-MH were proved to be stable under different storage and processing conditions. In addition, the detection was independent of matrix effect.This rapid and specific UPLC/MS-MS method is suitable for the determination of urinary 1-MH and 3-MH.