Tyrosine phosphorylation plays a critical role in cell biological functions, particularly in signal transduction. Mass spectrometry (MS)-based proteomic analysis of tyrosine-phosphorylated (pTyr) proteins  remains challenging due to their relatively low abundance. The methods of phosphopeptide enrichment are developed prosperously such as IMmuno Affinity hromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC). However, the complexity of experimental design for sample separation induces the unnecessary variations, particularly in label-free study designs. Here, we report an alternative approach, Inclusion List-driven Targeted LC tandem MS (ILT LC-tandem MS), and demonstrate the results  obtained  are comparable with that of phosphopeptide enrichment for increasing the sequencing possibility of low abundant pTyr peptides. In the present study, ILT LC-M/MS was applied for comparative label free quantitative tyrosine phosphor proteomic analysis between Src inhibitor (dasatinib)-treated and untreated CL1-5 lung cancer cells. A total of 512 distinct pTyr sites corresponding to 491 peptides had been identified. According to evaluation using Mascot Delta score (MD score), that 88.8% of the pTyr sites were considered confidently determined. In addition, the possibility of identifying multi phosphorylated peptides by ILT LC- tandem MS was 11% higher than that by TiO2-based phosphopeptide enrichment previously reported. After the comparative label-free quantitative analysis, among the 491 pTyr peptides identified 103 showed the significant level alteration of tyrosine phosphorylation between dasatinib-treated and untreated cells, and among which 53 altered pTyr peptides were matched to contain Src kinase substrate motifs. Using pathway analysis of the corresponding proteins, the results illustrated that 17 proteins were regulated by E2F transcription factor 1, which is known to be related to cell cycle regulation, and therefore provided a credible basis for further investigations. Our data demonstrate that the utility of an alternative approach, ILT LC--tandem MS, increases the sequencing possibility of low abundant pTyr peptides and reduces the induced variations in conventional TiO2 enrichment label-free quantitative analysis.