In this study, S-aminoethylation of Cys (AE-Cys) was carried out with 2-bromoethylamine to form pseudo-Lys, which was then digested with Lys-C or trypsin to improve proteome coverage in bottom-up proteomics. A model study with bovine serum albumin showed that the C-terminal side of Cys was successfully cleaved by AE-Cys followed by digestion with Lys-C. The frequency of side reactions to amino acids other than Cys was not as high as that of carbamidomethylation of Cys with iodoacetamide. Proteomic analysis of A549 cell extracts using AE-Cys identified more protein groups, especially membrane proteins, in a data-dependent acquisition mode. In addition, label-free quantification of mouse non-small cell lung cancer (NSCLC) tissue in data-independent acquisition mode showed improved NSCLC pathway coverage and higher reproducibility with AE-Cys. Furthermore, we successfully identified peptides containing the [T790M] mutation site of EGFR by AE-Cys, which has not been reported by conventional bottom-up methods despite its importance as one of the most important lung cancer-related mutation sites.