In this project, we developed a dual-functional data acquisition method, IsoPS-DIA, which incorporates two complementary designs: target windows and a non-target window. First, we evaluated the sensitivity and quantitative performance of targeted peptides, as well as the global proteomic profiling capabilities of IsoPS-DIA, in comparison with conventional fixed-window DIA (Fix-DIA), variable-window DIA based on precursor density (Var-DIA), and PRM. These comparisons were conducted using normal mouse lung tissue peptide samples spiked with targeted peptides, resulting in a total of 14 MS raw files and one searched result for each MS method. Next, IsoPS-DIA was applied to analyze six NSCLC cell lines—A549, PC9, H1975, H3255, CL68, and CL97—each harboring a distinct EGFR mutation (Wild type, Del19, L858R/T790M, L858R, Del19/T790M, and G719A, respectively). Samples were digested with Lys-C, and each cell line was analyzed in triplicate, yielding a total of 18 MS raw files and one Spectronaut searched result. Additionally, to generate Del19 peptides, Glu-C digestion was performed on PC9 and CL68, resulting in six more MS raw files. We also provide the result of IsoPS-DIA performed in Q-TOF instruments (40 MS raw files). In total, 124 raw files and their corresponding searched results were uploaded.