Abstract Treatment options available for cancer patients are increasing rapidly. Biomarkers to predict which therapies will be effective are however lacking, resulting in the frequent administration of ineffective therapies. Emergence of therapy resistance and both inter-patient and intra-patient heterogeneity play an important role in the differences in response to treatment in prostate cancer (PC). Therefore, research into the nature of the heterogeneity and its significance will aid in developing an improved individualized treatment strategy. Here we use a microwell technology that can enable the testing of drug effectivity and heterogeneity of tumor cells. Several techniques exist to study secreted proteins, including enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISpot), intracellular staining, and cytokine capture. The common immunoassays that directly measure secreted proteins on a solid support (ELISA and ELISpot) fail to correlate the captured analytes with the producing cell and cannot do this repeatedly on the same cell. We used the microwell technology to sort viable single cells derived from the prostate cancer cell lines LnCAP and VcCAP. The Prostate Specific Antigen (PSA) produced by LnCAP and VcAP cells is measured with or without drug stimulation (r1881, Enzalutamide or Abiraterone). To measure PSA secretion the microwell is connected to a membrane coated with antibodies against PSA. We demonstrate that PSA secreted by cells can be measured and we quantified the amounts for >1200 single PC cells. Before cells were subjected to drug stimulation PSA secretion was measured. Results indicate a heterogeneous PSA secretion profile for both cell lines; the average LnCAP secretion patterns were 4,7 (sd=3,3), 11,3 (sd=5,9) and 2,4 (sd=1,1) pg/cell for respectively, non-stimulated, r1881 androgen steroid and Enzalutamide respectively. LnCAPs stimulated with Abiraterone secreted 4,7 (sd=1,9) pg/cell, indicating that enzalutamide has a higher inhibitory effect on PSA secretion than abiraterone. VcAPs secreted 3,8 (sd=1,8), 6,5 (sd=4,1) and 0,9 (sd=0,7) pg/cell for non-stimulated, r1881 androgen steroid and enzalutamide stimulated cells respectively. Abiraterone stimulated cells secreted 2,0 (sd=1,5) pg/cell. The results further suggest that a small percentage of LnCAP of the cells (<1%) did not respond to Enzalutamide treatment indicating resistance to the drug. We expect that this approach can enable to study and predict the efficacy of the clinical response to drug and ultimately improve the success rate of treatment of PC patients. This research is part of the HTSM research programme and was partly funded by the NWO, The Netherlands Organization for Scientific Research (Utrecht, The Netherlands) and is part of Project McSPRinter under project number 15327. Citation Format: Fikri Abali, Narghes Baghi, Arjan Tibbe, Leon Terstappen. Detection of PSA secretion from single prostate cancer cells with and without drug stimulation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2693.