A binding enzyme-linked immunosorbent assay (ELISA) has been developed for measuring nanogram concentrations of semisynthetic pneumocandin antifungal agents in human plasma. Semisynthetic pneumocandin L-733,560 was conjugated to succinylated hemocyanin by water-soluble carbodiimide and was used as an immunogen to produce polyclonal antibodies in rabbits. Pneumocandins were used to directly coat the wells of a microtiter plate, and quantitation was achieved by using rabbit polyclonal antibodies to pneumocandin L-733,560 and goat anti-rabbit immunoglobulin G conjugated to either alkaline phosphatase or horseradish peroxidase. Maximum binding of L-733,560 and most related analogs to the wells of the microtiter plate was found to occur in the first 5 min of incubation at 4°C. Once bound to the plate, these pneumocandins could not be removed from the plate, either by treatment with 4.0 to 6.0 M urea or by treatment with 4.0 to 6.0 M guanidine hydrochloride for 24 h at 4°C. The binding ELISA is linear with drug concentration and can detect levels of L-733,560 as low as 5 ng/ml in human plasma. The assay is also useful for quantitating plasma levels of related semisynthetic pneumocandins including clinical candidate MK-0991.