Colorectal cancer is one of the most common cancer types worldwide and it continues to be a serious public health problem. Early detection and diagnosis are of great importance in cancer management. At present, diagnostic blood tests are based on the detection of tumor-associated markers such as carcino-embryonic antigen (CEA), the cancer antigen CA19-9 for gastrointestinal cancer, CA15-3 for breast cancer or CA125 for ovarian cancer. The lack of sensitivity and specificity of these markers prevents their general use in cancer screening of an average risk population. Therefore, new cancer biomarkers or better screening methods are necessary to improve the diagnostics of the disease. This study was directed to the optimization of a diagnostic, enzyme linked immunosorbent assay (ELISA) based test to identify and validate new serum markers, such as extracellular Protein Kinase A (ecPKA) and Nicotinamide N-Methyltransferase (NNMT). In this type of assay, the cancer antigens are quantified indirectly - by detecting the presence of auto-antibodies against tumor proteins in human serum. The result of the optimization and validation process was in the case of ecPKA a reproducible and stable assay. In case of NNMT the assay was probably not sensitive enough.