An enzyme-linked immunosorbent assay (ELISA) for the determination of anti-ricin immunoglobulin G (IgG) concentration in mouse sera was systematically validated. The results obtained throughout the validation process strongly demonstrated that the ELISA was reliable, reproducible, and suitable for its intended use. The assay had a high level of precision within and between runs, was specific for the anti-ricin IgG, and showed no interference with a number of different serum matrices. The assay exhibited excellent accuracy, linearity, and stability. The mean recovery of four test samples with different known concentrations was 100.9+/-11.3%, 102.7+/-10.8%, 99.0+/-7.2%, and 95.9+/-11.3%, respectively (n=10). The mean recovery of the observed anti-ricin IgG concentration of three quality control samples run on 73 plates to their nominal concentrations was 100.1+/-7.3%, 100.2+/-5.8%, and 103.7+/-8.1%; and the coefficient of variation (CV) was 7.3%, 5.8%, and 7.8%, respectively. The back-calculated anti-ricin IgG concentration, %CV, and relative error of seven standards from the calibration curves run in the entire validation study were analyzed (n=7 x 73). The results indicated that the four-parameter logistic (4PL) equation, y=(a-d)/(1+(x/c)b)+d, provided an accurate representation of a sigmoidal relationship between the measured response and the logarithm of observed concentration of anti-ricin IgG in mouse sera for this ELISA. The lower limit of quantification and upper limit of quantification of the calibration curve were 3.3 ng/ml and 82.8 ng/ml, respectively. The measurable range of the assay would cover all possible anti-ricin IgG concentrations in mouse sera stimulated with a ricin vaccine candidate, when the test sera are measured at a 1:800 starting dilution followed by four additional fourfold serial dilutions.