Despite significant advances in the treatment of cardiovascular diseases, antiplatelet therapies are still associated with a high risk of hemorrhage. In order to develop new drugs, methods to measure platelet function must be adapted for the high-throughput screening (HTS) format. Currently, all assays capable of assessing platelet function are either expensive, complex, or not validated, which makes them unsuitable for drug discovery. Here, we propose a simple, low-cost, and high-throughput-compatible platelet function assay, validated for the 384-well plate. In the proposed assay, agonist-induced platelet activity was assessed by three different methods: (i) measurement of light absorbance, which decreases with platelet aggregation; (ii) luminescence measurement, based on ATP release from activated platelets and luciferin-luciferase reaction; and (iii) automated bright-field microscopy of the wells and further quantification of platelet image area, described here for the first time. Brightfield imaging results were validated by demonstrating the similarity of dose-response curves obtained with absorbance and luminescence measurements after stimulating platelets, pre-incubated with prostaglandin E1 or tirofiban, and demonstrating the similarity of dose-response curves obtained with agonists. Assay quality was confirmed using the Z'-factor, a statistical parameter used to validate the robustness and suitability of an HTS assay. The results showed that, under high rotations per minute (1200 RPM), an acceptable Z'-factor score is reached for absorbance measurements (Z'-factor - 0.58) and automated brightfield imaging (Z'-factor - 0.52), without the need of replicates, while triplicates must be used to achieve an acceptable Z'-factor score (0.54) for luminescence measurements. Using low platelet concentration (4 x 10(4)/l - 10 l), the brightfield imaging test was further validated using washed platelets. Furthermore, drug screening was performed with compounds selected by structure-based virtual screening. Taken together, this study presents an optimized and validated assay for HTS to be used as a tool for antiplatelet drug discovery.