Determination of gossypol in egg yolk samples was studied by using HPLC-ESI-MS/MS. The sample was extracted with acetone and analyzed on a C18 column using methanol and 1% formic acid as mobile phase. The monitoring ions of gossypol were 517.4/259.3 and 517.4/231.3 in the multiple reaction monitoring mode (MRM). Linear relationship between values of peak area and mass concentration of gossypol was obtained in the range of 1 ng/mL~100 ng/mL, with lower limit detection (10 S/N) of 1 ng/mL. Recovery test was made by addition of standard gossypol with different concentrations. The recovery was found in the range of 85%-100%. RSD was found to be less than 10% (n=6). The method was simple, sensitive, accurate and repeatable.