Here, we applied drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) approaches with non-labeled Erlotinib to explore possible binding proteins besides EGFR and discovered that DNA polymerase alpha subunit B (POLA2) positions as a new binding protein candidate, responsible for Erlotinib resistance in NSCLC. This evidence concerns the gene EGFR and non-small cell lung carcinoma.