Ascites tumor cells (ATCs) represent a potentially valuable source of cells for monitoring treatment of ovarian cancer as it would obviate the need for more invasive surgical biopsies. The ability to perform longitudinal testing of ascites in a point-of-care setting could significantly impact clinical trials, drug development, and clinical care. Here, we developed a microfluidic chip platform to enrich ATCs from highly heterogeneous peritoneal fluid and then perform molecular analyses on these cells. We evaluated 85 putative ovarian cancer protein markers and found that nearly two-thirds were either nonspecific for malignant disease or had low abundance. Using four of the most promising markers, we prospectively studied 47 patients (33 ovarian cancer and 14 control). We show that a marker set (ATCdx) can sensitively and specifically map ATC numbers and, through its reliable enrichment, facilitate additional treatment-response measurements related to proliferation, protein translation, or pathway inhibition.