OBJECTIVE： To improve the quality standard of Ejiao sanbao cream. METHODS： UPLC-MS was adopted to identify the donkey-hide glue in the preparation， the chromatographic conditions： column was Acquity UPLC BEH-C18 with mobile phase of acetonitrile-0.1％ formic acid （gradient elution）， volumetric flow rate was 0.3 mL/min，injection volume was 5 μL， ion source is electrospray ionization source， capillary voltage was 3.5 kV， capillary outlet voltage was 120 V， cone voltage was 50 V， desolvation temperature was 400 ℃， drying gas flow was 10 L/min， nebulizer pressure was 40 psi， collision energy was 10-45 V， scan range was m/z 200-1 500； detection mode was positive ion mode （ESI+），multiple reaction monitoring. HPLC was used to determine the contents of 4 hydrohyzing amino acid： column was Agela Venusil XBP-C18 with mobile phase A of acetonitrile-0.1 mol/L sodium acetate solution （36％ acetic acid adjusted to pH 6.5） （7 ∶ 93，V/V） and B of 80％ acetonitrile solution （gradient elution） at a flow rate of 1.0 mL/min， the detection wavelength was 254 nm， and column temperature was 43 ℃. RESULTS： The UPLC-MS peak of Equus asinus was clear and specific， without negative interference. The linear range was 0.081 9-0.655 2 μg for L-hydroxyproline （r＝0.999 9）， 0.186 2-1.489 6 μg for glycine（r＝0.999 8）， 0.070 3-0.562 0 μg for alanine （r＝0.999 9） and 0.124 2-0.993 6 μg for L-proline（r＝0.999 9）； RSDs of precision， stability and reproducibility tests were lower than 2.0％； recoveries were 99.85％-103.69％ （RSD＝1.35％，n＝6）， 99.91％-103.93％（RSD＝1.46％，n＝6）， 96.86％-101.27％（RSD＝1.69％，n＝6） and 97.44％-101.45％（RSD＝1.54％，n＝6）， respectively. CONCLUSIONS： The improved standard can more effectively control the quality of Ejiao sanbao cream.