The viability and interpretation of techniques for the evaluation of immunocompetence of animals in their natural environment has been largely debated. One of these methods is based on testing the antimicrobial capacity of the blood and/or plasma in vitro, which could rapidly and effectively assess the immunological conditions of natural populations. We tested the applicability of the antimicrobial capacity of plasma (ACP) assay in anuran amphibians from the Atlantic Forest. The assay was performed by measuring both the turbidity (in a spectrophotometer) and the colony forming units (CFU) of the remaining bacteria (Escherichia coli) following exposure to amphibian plasma. Although both assays were correlated, the ACP assay by spectrophotometry showed 10 times lower intra-assay variation. We also found interspecific variation in ACP, as well as the maintenance of ACP values in males from the same population, collected in different breeding seasons. Thus, the estimation of ACP by spectrophotometry provides a convenient and accurate method for evaluating innate immunocompetence in comparative and ecophysiological studies of anuran amphibians.