To determine residual acrylamide in medical polyacrylamide hydrogel by high performance liquid chromatography tandem mass spectroscopy (HPLC-MS). After 13 C 3 labeled acrylamide was added, the sample was extracted with water and then cleaned up with Extrelut TM 20. The polyacrylamide hydrogel sample and 20 clinical cases were analyzed by HPLC-MS/MS and isotope dilution quantifying technique in selected reaction monitoring (SRM) mode. Acrylamide was separated from polyacrylamide hydrogel. The concentration of acrylamide in polyacrylamide hydrogel ranged from 3.9×10 −9 to 3.1×10 −8 g/L in the 20 clinical cases. The peak area was favorable linear and the range was up to 3 000 μg/L. The recovery rate was 103.1% with a relative standard deviation (RSD) of 6.20%, when the mark level was 50 μg/L. HPLC-MS is a rapid, accurate, and sensitive method for the determination of residual acrylamide in medical polyacrylamide hydrogel.