Aberrant activation of splicing regulators contributes to oncogenic transformation of cells by modulating the alternative splicing events of key oncogenes. Phosphorylation of splicing factors affects subcellular localization and activity of these proteins. Studies have shown a close association between altered phosphorylation of molecules involved in splicing machinery and cancer. We carried out proteomic and phosphoproteomic analysis of head and neck squamous cell carcinoma (HNSCC) cell lines using tandem mass tag (TMT) labeling approach followed by titanium dioxide-based phosphopeptides enrichment method. LC-MS/MS analysis resulted in the identification of 4,920 phosphosites corresponding to 2,288 proteins in six HNSCC cell lines compared to a normal oral cell line; OKF6/TERT1. Among the proteins identified, 23 kinases were found to be hyperphosphorylated in all HNSCC cell lines compared to the normal oral cell line.