We describe an enzyme-linked immunosorbent assay (ELISA) to quantitate venom antigens in human serum and urine, and thus to help evaluate the severity of envenomation due to viper bites. This assay, which is performed with commercially available polyclonal Fab'2s in a double-sandwich method, is rapid, simple, and specific for antigens of European vipers (Vipera aspis, Vipera berus, and Vipera ammodytes). No cross-reactivity was observed with other snake venoms or human serum proteins. It showed a good linear response over a wide range of concentrations of venom antigens (from 1 to 100 ng/mL). It was very sensitive, with detection limits of 7 and 2 ng/mL for Vipera aspis venom in serum and urine, respectively. This ELISA is also easily reproducible; the coefficients of variation determined at different concentrations of venom (50, 25, and 5 ng/mL) did not exceed 10% in serum and 14% in urine samples collected from different donors. This test was applied to determine the concentrations of venom in the serum of patients bitten by a viper in France and to follow its elimination as a function of time. The method is adaptable to other venoms by using other specific immunoglobulins.