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In the present study an ELISA assay was developed and validated for detection and determination of the concentration of snakes venom in biological samples. Individual component of each venom (Cerastes cerastes and Macrovipera mauretanica) used as immunogen to raise specific rabbit IgGs in order to set up a sandwich-type ELISA. Lower detection limit, linearity, accuracy, precision, reproducibility, and reference intervals were determined. The method proved to be simple, specific, reproducible, sensitive (detection limit = 0.5 ng/ml) and the calibration plot was based on linear regression analysis (r = 0.980) between 0.9 and 1000 ng/mL of venom concentration, with a lower limit of quantification of 1.58 ng/mL. The intra- and interassay coefficient of variation ranged from 2,02 to 4.62% and 5.29 to 7.40%, respectively. The specificity of the assay was tested using vipers, cobra and scorpion venom. This method detected venom from all viper species tested without significant cross reactivity with other venoms in the concentration range of 0.9–1000 ng/mL. This ELISA described is sufficiently validated for clinical evaluation. The method is adaptable to other venoms. This is potentially useful for clinical diagnosis of snakebite, to monitor antivenom dose, and consequently to improve the national health monitoring systems.