Objective To develop a size exclusion-high-performance liquid chromatography(SEC-HPLC) method for the simultaneous determination of free proteins and residual 4-dimethylaminopyridine(DMAP) in the bulk solution of pneumococcal polysaccharide-protein conjugate, and to verify and preliminarily apply the method.Methods By studying the characteristic ultraviolet absorption peak of DMAP and optimizing the separation method of chromatographic column, the SEC-HPLC method was established to realize the retention and separation of free proteins and DMAP on the same chromatographic column and quantify them. The method was verified for specificity(system suitability), limit of quantification(LOQ), accuracy, precision, linearity and range, and robustness, and was used to detect the free proteins and DMAP residues in the test sample.Results The chromatographic conditions were determined as follows: TSKgel G5000 PWXL analytical column was used as the column(7. 8 mm × 300 mm, 10 ??m), the column temperature was set at 35 ℃, the mobile phase was0. 85% sodium chloride solution, the pH was(6. 2 ± 0. 2), the flow rate was 0. 5 mL/min, and the detection wavelength was280 nm. The resolution of free protein peaks of diphtheria toxoid(DT) or tetanus toxoid(TT) and DMAP peak from adjacent peaks was greater than 1. 5. The LOQs of TT, DT and DMAP were 0. 50, 1. 20, and 0. 02 ??g/mL, respectively. The spiked recovery rates of TT, DT and DMAP were 98. 11%-104. 60%, 95. 43%-103. 26% and 97. 11%-98. 42%, respectively.The relative standard deviations(RSDs) of intermediate precision of TT, DT and DMAP were all less than 5%. The R~2 values of TT and DT were greater than 0. 999 0 in the range of 6-24 ??g/mL, and the R~2 value of DMAP was 1. 000 in the range of2-10 ??g/mL. The RSDs of TT, DT and DMAP content were all less than 3% at the injection temperature of 30, 35 and 40 ℃.The established method was used to determine the content of free proteins and DMAP in the test sample, and rapid and accurate results were obtained.Conclusion The established SEC-HPLC method has good system suitability, accuracy,precision, linear relationship and robustness, which can realize the dual quantification of free proteins and DMAP in the same experiment, and can be used to monitor the impurities in the binding process.