Here we demonstrate the utility of high-density antibody microarrays for ovarian cancer biomarker discovery. This report describes the technology and how it can be optimized for hypothesis-generating and testing experiments. Our previous results validated the high density antibody array technology platform, the current work expands on it utilizing a second generation array that we tested with a larger set of ovarian case and control serum samples. We then describe our strategies and methods for result validation, including Western immunoblots to confirm antibody specificity. By comparing and combining the current results with our previous study, we solidified the case that the markers found could be used for ovarian cancer diagnosis using this technology. These results set the stage for further validation of these potential biomarkers and the use of this technology in future biomarker discovery studies.