Proteomics one of the omics methods that investigates the functions, structures and expression of all proteins in a biological system. It is important for understanding the disease mechanisms and brings together some innovative techniques such as mass spectrometry, Western blotting and electrophoresis. The DARTS method (Drug affinity responsive target stability) is the name of a set of proteomics techniques used to investigate the biological targets of drugs. The principle of its work is based on the fact that proteins better resist the digestion of proteolytic enzymes if a ligand (drug) is attached to them. It has a special role in the research of unknown targets of small molecules that have the ability to aim at specific proteins important for regulating intracellular signaling pathways. Samples of cell lysates are treated with a small molecule, subjected to protease digestion and then analyzed qualitatively and quantitatively. A special advantage of DARTS is that it uses the drug in its native form, without any modification. This method is used for different types of research, and mostly for research into known and unknown targets of drugs, the mechanisms of action of drugs, but also the mechanisms of resistance. The small molecule chosen in this thesis is erlotinib (Tarceva), whose known biological target is the Epidermal growth factor receptor (EGFR). Erlotinib is a tyrosine-kinase inhibitor which stops the proliferation of tumor cells and the avoidance of apoptosis when bound. Samples for the DARTS method were prepared using lysates of HeLa and A431 cells that overexpress EGFR. Pronase was the protease enzyme used for digestion, and proteins were separated using gradient SDS-PAGE electrophoresis, according to molecular weight. Western blotting confirmed the specificity using antibodies and increased the sensitivity of the method, which resulted in a better visualization of the change in the intensity of EGFR, to which erlotinib is bound. It was confirmed that EGFR is a direct target of erlotinib, but also that its mechanism of action is stopping the autophosphorylation that interrupts the signaling pathway for cell proliferation. Unknown targets of erlotinib (off-targets), that show a significant change in intensity in its presence, were also investigated, but not identified. It was concluded that DARTS is a multipurpose, fast and simple biophysical method that does not require chemical modifications of the drug and is useful in many aspects of drug research and development.