In this study, a rapid and reliable HPLC-MS/MS method for the determination of 20 PFAs in animal liver was developed by comparing the extraction efficiency of different extractants and optimizing the extraction and purification conditions. The samples were quantified using isotope internal standard and external standard with the matrix matched standard calibration curve method. The results indicated that this method was simple, rapid, sensitive, and accurate. The method can be widely used for the determination of PFAs in animal samples due to its excellent advantages such as less solvent consumption and effective correction of matrix effect.