Currently only a limited number of tumor markers for non-small cell lung cancer (NSCLC) are available. Antibodies to tumor-associated proteins may expand the number of available tumor markers for lung cancer and be used together in a serum profile to enhance sensitivity and specificity. In this study, we isolated 57 tumor-associated proteins from two NSCLC cDNA T7 phage libraries using biopan enrichment techniques with NSCLC patient and normal sera. Sequence analysis showed that among the 57 phage-displayed proteins 45 have sequence identity with known or putative tumor-associated proteins. Immunochemical reactivity of patient sera with phage-expressed proteins showed enrichment on the number of immunogenic phage clones in the biopanning process and also confirmed that antibodies were present in patient sera but not in normal sera. Antibodies to five phage-expressed proteins were measured by enzyme-linked immunosorbent assay (ELISA) to validate the concept that combinations have greater predictive value than any single antibody alone. Logistic regression analysis showed that combined measurements of five antibodies was more predictive of disease than any single antibody alone, underscoring the importance of identifying multiple potential markers. The resulting antibody profiling is a feasible diagnostic strategy for NSCLC. An inventory of corresponding proteins may have significant relevance to tumor biology, novel drug development, and immunotherapies.