ATP-analogue inhibitors, Gefitinib (Iressa) and Erlotinib (Tarceva), had been approved for advanced and metastatic NSCLC against tyrosine kinase domain of EGFR. Many techniques have been developed to better understand the drug mechanism which is multistep, time-consuming, and expensive. Herein, we performed Fourier transform infrared microscopy (FTIR) for evaluating the biochemical change on NSCLC (A549) cells after treatment. At levels that produced equivalent effects, Gefitinib dramatically induced cell apoptosis via impaired mitochondrial transmembrane potential. Whereas, Erlotinib had a slight effect on A549. Principal component analysis (PCA) was performed to distinguish the effect of EGFR inhibitors on A549. FTIR spectra regions were divided into three regions; lipids (3000-2800 cm-1), proteins (1700-1500 cm-1), and carbohydrates and nuclei acids (1200-1000 cm-1). Biochemical changes can be evaluated by these spectral regions. This work may be a novel concept for utilizing FTIR spectroscopy for high-throughput discriminative effects of a drug or compound and its derivatives on cells. This article is protected by copyright. All rights reserved.