It is a challenging task to verify and quantify potential biomarkers expressed at elevated levels in sera from cancer patients. An immunoaffinity-mass spectrometry based approach has been developed using antibodies to enrich proteins of interest from sera followed by mass spectrometry based quantification. Antibodies specific to the protein of interest were immobilized to hydrazide resin via the carbohydrate moiety on the Fc region of the antibody. Captured proteins were eluted, reduced, alkylated and digested with trypsin. Peptides were analyzed by liquid chromatography coupled with multiple reaction monitoring (LC-MRM) approach and quantification was achieved by the addition of stable isotope labeled (heavy) standard peptides. Using this methodology, we were able to achieve a linear response from 15 ng/ml to 250 ng/ml for CEA, a known tumor biomarker. Moreover, we have observed elevated levels of CEA in sera samples from lung cancer patients which to our knowledge is the first time that circulating CEA has been detected by mass spectrometry-based analysis. This approach was further applied to potential protein biomarkers discovered from tumor cell lines and tumor tissues. Linear response was obtained from a multiplex spiking experiment for SLPI (4 – 500 ng/ml), TFPI (42 – 1000 ng/ml), TFPI2 (2 – 250 ng/ml) and TIMP1 (430 – 1000 ng/ml). A replicate experiment for a single concentration value yielded a relative CV better than 11% for TFPI, SLPI and TFPI2. The expression level of the proteins in lung cancer patient sera was assayed by immunoaffinity-MRM method and the results were comparable with those obtained from ELISA. This immunoaffinity-mass spectrometry based quantification approach thus provides a specific and accurate assay for verifying the expression of potential biomarkers in patient serum samples, especially for those proteins for which the necessary reagents for ELISA development are unavailable.