Abstract: The simultaneous determination of rhamnose, ribose, arabinose, fucose, galactose, xylose, galacturonic acid, mannitol, glucuronic acid and mannose in whey protein sports nutrition powder polysaccharide hydrolyzate was established. An ultra-high performance liquid chromatography-tandem mass spectrometry method was used for the analysis of 12 sugar compounds. The samples of whey protein sports nutrition powder were extracted by ultrasound, and the protein was removed by the Sevag method until no protein was precipitated, and then the monosaccharide composition was determined by acid hydrolysis. Using 5 mmol/L ammonium acetate+0.02% formic acid aqueous solution as mobile phase A, methanol as mobile phase B, in the electrospray ion source negative ion (electrospray ion source negative ion, ESI⁻), in the multiple reaction monitoring mode (multiple reaction monitoring, MRM) analysis, the detection was conducted by external standard method for quantification. The results showed that in the mass concentration range of 0.10~6.00 μg/mL, the 12 sugar compounds had good linear relationship with each monosaccharide, the correlation coefficients (r) were all greater than 0.995. The detection limit was 0.0037~0.0326 mg/g. The quantitative limit was 0.0123~0.1087 mg/g. 12 sugar compounds were added to the samples at three levels with concentrations of 40, 100 and 160 mg/g, and the average recovery was 76.12%~99.12%, relative standard deviations (RSDs) 0.12%~10.01% (n=6). This method was used to analyze the samples of whey protein sports nutrition powder, and the results showed that 12 carbohydrate compounds were detected in different degrees in 15 samples, and the content of various monosaccharides in different samples was quite different. The establishment of this method could provide technical support and basic data for the structural composition and activity of whey protein sports nutrition powder polysaccharide.