Updated project metadata. Mass spectrometry is a rational orthogonal method for antibody-based assays, but implementation of MS in the validation pipeline of antibody manufacturers is hampered by the high cost and low throughput. Here we present a rapid method for antibody validation based on denaturing gel electrophoresis of biotinylated cell lysates (PAGE) followed by mass spectrometry (MS) and antibody array analysis (MAP). The first step, PAGE, produces 12 fractions containing proteins of increasing molecular weight. The fractions are analyzed in parallel by MS and MAP. Antibodies to be tested are immobilized on color coded polymer beads to create antibody arrays, which can comprise up to several thousand various antibodies. MS data provide definite protein identifications in each fraction, creating a reference for antibody reactivity patterns obtained via MAP. The method employs automated software to compare both datasets and provide validation data for each antibody tested. Due to the high-throughput nature of the assay we were able to screen several thousands of antibodies against six different cell lines. The differences in protein expression between the cell lines provide an additional control of antibody specificity. Using PAGE-MAP it is possible to screen and validate thousands of antibodies in a matter of weeks. Moreover, antibodies are tested under standardized conditions, which allows for direct comparison of their performance.