A label-free approach to qualify and quantify bovine serum albumin（BSA） based on the information of mass spectrum was reported.The conditions of high-performance liquid chromatography（HPLC） were optimized,including program of gradient elution and flow rate.The content of BSA was determined in optimal conditions by HPLC-quadrupole-time of flight tandem mass spectrometry（Q-TOF）after digested with trypsin in-solution.The data was processed with MassHunter Qualitative Analysis software.The setting parameters of software were optimized to decrease the probability of false positive results.The reproducibility of the experiments was investigated.According to the peak area results of eight peptides,the RSDs（n=4） were below 3.0%.The detection limit for BSA was achieved as 100 ng.The results also indicated that there was a logarithmic manner between concentration of protein and sequence coverage of peptides.Among the eight peptides of BSA with higher abundance,one unique peptide:HLVDEPQNLIK was selected to qualify the BSA,and another unique peptide:ATEEQLK was used to quantify the BSA.The recovery of this method was calculated to be 106%.This method is simple,rapid,low cost and specific,and could be employed for screening characteristic peptides of specific proteins and detecting of complex biological sample.