Tumor-associated antigens (TAAs) can be used as cancer markers and as signposts of therapeutic targets since their inimitable expression in cancer or significant overexpression in esophageal squamous cell carcinoma (ESCC) correlates with the initiation and progression of the diseases. Immunoblotting, also known as Western blotting or protein blotting, is a core technique in cell and molecular biology to detect proteins and glycoproteins. The technique allows detection of TAAs from complex protein samples such as in serum, aspirate, or solid tumor homogenate. In the process, proteins are separated according to the molecular weight. They were visualized within a gel matrix and then transferred to a supporting membrane. Finally, they are probed for binding with corresponding antibodies and identified the target proteins. Herein, we describe the Western blots analysis to detect protein or glycoprotein in samples from patients with esophageal squamous cell carcinoma (ESCC) or cells derived from ESCC.