Aim: To support pharmacokinetic studies and therapeutic monitoring of darunavir (DRV), a highly sensitive ELISA was developed for the determination of DRV in plasma samples at picogram levels. Results: The assay LOD and LOQ were 15 and 30 pg ml^-1, respectively. The working range of the assay was 20–2000 pg ml^-1. Analytical recoveries of DRV from spiked plasma were in the ranges of 98.4–113.0 and 86.0–99.1% for intra-assay and inter-assay runs, respectively. The precision of the assay was satisfactory. Conclusion: The ELISA is characterized by high throughput and it is expected to significantly contribute to routine analysis of DRV in its pharmacokinetic studies and therapeutic monitoring.