Pokeweed antiviral protein (PAP) is a soluble leaf protein produced by the plant Phytolacca americana. It is able to specifically remove an adenine residue from the 28 S ribosomal RNA of eukaryotic ribosomes, thus blocking protein synthesis. Although PAP seems to be synthesized constitutively at very high concentrations during plant development, the mechanism of its synthesis remains unclear since the protein is toxic to pokeweed ribosomes. Recently, our group identified an inactive complexed form of PAP (PAPi) which could play a significant role in the plant protection mechanism during PAP synthesis. In order to estimate the concentration of PAP in plant extracts, a double sandwich ELISA was developed. This test enabled the detection and the quantification of PAP in a crude leaf extract at concentrations ranging from 2–1000 ng ml −1. It was specifically developed to discriminate between free PAP and PAPi. This report shows that the use of a monoclonal antibody specific to the C-terminal extremity of mature PAP, combined with an anti-PAP polyclonal antibody did not detect PAPi, unless it was denaturated prior to ELISA. Thus, the specificity of this test was restricted to free PAP and the test proved to be a reliable and unique tool to estimate the integrity of PAPi.