With the reintroduction of nonruminant processed animal proteins (PAPs) for use in aquaculture in 2013, there is a suitable alternative to replace expensive fish meal in fish feed. Nevertheless, since the bovine spongiform encephalopathy (BSE) crisis, the use of PAPs in feed is strictly regulated. To date, light microscopy and polymerase chain reaction are the official methods for proving the absence of illegal PAPs in feed. Due to their limitations, alternative methods for the quantitative species differentiation are needed. To address this issue, we developed and validated an 8-plex mass spectrometry-based immunoassay. The workflow comprises a tryptic digestion of PAPs and blood products in suspension, a cross-species immunoaffinity enrichment of 8 species-specific alpha-2-macroglobulin peptides using a group-specific antibody, and a subsequent analysis by ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry for species identification and quantification. This workflow can be used to quantitatively determine the species origin in future feed authentication studies.