Objective  To develop and verify a high performance liquid chromatography (HPLC) method for determination of residual Triton X-100 content in quadrivalent influenza virus split vaccine (MDCK cells). Methods  Triton X-100 residue in the vaccine was detected by HPLC, and the system applicability, specificity, linearity, accuracy, precision, durability, quantification limit, and detection limit of this method were verified. Results  The numbers of theoretical plates of Triton X-100 detected by HPLC were 1 560-1 599. The relative standard deviations (RSDs) of peak area and retention time of Triton X-100 were 1.93% and 0.30%, respectively. The Triton X-100 chromatographic peak and impurity separation was good. No interference peak appeared in the blank solution. In the range of 5-160 μg/ml, the Triton X-100 concentration showed good linear relationship to the peak area (coefficient of determination at 0.999). The spike recovery rates of Triton X-100 at various concentrations were 95%-105%, all with RSDs no more than 2%. The RSDs of peak area of 6 samples tasted by one staff was 0.58%, and the RSDs of peak area by one staff on various working days and by various staff on one working day were all less than 2%. After the change of column temperature, the RSD of peak area was 0.45%. The limits of detection and quantitation were 1 and 5 μg/ml, respectively. Conclusions  HPLC detection method for Triton X-100 residue in quadrivalent influenza virus split vaccine is successfully established. The system applicability, specificity, linearity, accuracy, precision, and durability of this method are good.