Objective: To establish a high performance liquid chromatography (HPLC) method for the simultaneous determination of principal components in protamine sulfate, and to employ LC-MS for the identification for the amino acids sequences of main peptides. Methods: The ThermoHypersil GOLD column (250 mm×4.6 mm, 5 μm) was selected for analysis with a gradient program of mobile phase A[0.1 mol·L^-1 sodium dihydrogen phosphate solution(adjusted to pH 1.8 with phosphoric acid)]and B[acetonitrile-0.1 mol·L^-1 sodium dihydrogen phosphate solution(adjusted to pH 1.8 with phosphoric acid) (6.5∶93.5, v/v)].The column temperature was 55 ℃. The UV detection wavelength was 214 nm and the flow rate was 1.0 mL·min^-1. The injection volume was 100 μL. The LC-MS method adopted distillate collection method to extract and enrich each peptide, Waters ACQUITY UPLC Peptide BEH C₁₈ column (15 cm×0.21 cm, 1.7 μm) was selected for liquid-mass spectrometry method, flow rate was 0.2 mL·min^-1, the mobile phase A was 0.1% formic acid solution, mobile phase B was 5% acetonitrile-0.1% formic acid solution, gradient elution. Thermo Q-Exactive Plus, ESI ion source, cation detection mode, atomization voltage was 3 800 V, ion transfer tube temperature was 300 ℃, scanning range of m/z 200-2 000. Results: The HPLC method established in this paper could identify the protamine sulfate from salmon and herring provided by the two companies. The content of protamine sulfate was 94.4%-96.2% from salmon, and 88.4%-92.3% from herring, protamine sulfate purity determination result was 96% from salmon, and 66%-69% from herring. The amino acid sequence of the target peptide of protamine was retrieved and obtained from Uniprot protein database, and analyzed by ThermoBioPharmaFinder v3.0 software to map fragments ion peaks were matched with theoretical fragment ions of the target sequence, and the amino acid sequence identification results of 4 main peptides from salmon and 3 main peptides from herring were obtained. Conclusion: The method established in this paper can be used for the identification, content determination and purity analysis of protamine sulfate. The LC-MS method can be used for the identification of protamine sulfatepeptide sequences from salmon and herring.