Protein termini play pivotal roles in various biological processes. Although several terminomic strategies have been proposed for the analysis of protein N-termini or protein C-termini separately, few can analyze both ends of proteins at the same time. Herein, we developed a workflow, termed Simultaneous Analysis of Protein N- and C-Terminome (SAPT) based on strong cation exchange chromatography (SCX) fractionation. Taking advantage of terminal peptides' reduced charge states in low pH SCX for their selective separation, we identified 3724 canonical human protein N-termini and 3129 canonical human protein C-termini, as well as 1463 neo-N-termini from the HeLa cell line, representing the largest human protein C-termini data set and the second largest human protein N-termini data set so far. The whole fractionation procedure was simple and rapid with considerable selectivity by utilizing a commercially available SCX-SPE column. In addition, we report for the first time the comprehensive screening of protein N-terminal and C-terminal modifications, leading to an identification of 8 kinds of protein N-terminal PTMs other than acetylation and 1 kind of protein C-terminal PTM other than amidation. Our results demonstrate the excellent performance and great potential of SAPT in terminomic studies. Data are available via ProteomeXchange with identifier PXD024573.