Due to a growing aquaculture industry, demand for high-quality proteins for aquatic feeds is increasing. Non-ruminant processed animal proteins (PAPs) have shown great potential for this purpose. Safe reintroduction of non-ruminant PAPs in aqua feed requires methods that can discriminate ruminant and non-ruminant PAPs at contamination levels at or below 2%. Because the official European Union method lacks species specificity, the performance of MELISA-TEK™ Ruminant, a commercial immunoassay, combined with the MELISA-TEK High Sensitivity Sample Extraction kit was evaluated. Various non-ruminant PAPs spiked with ruminant PAPs (processed at 133, 137, 141, and 145°C) were analyzed. Results showed an overall specificity of 99%, indicating no cross-reaction with non-ruminant PAPs. The sensitivity of the assay strongly depended on both processing temperature and proportion of muscle fibers of the ruminant PAPs. Overall sensitivity of samples with 1 and 2% ruminant PAPs was 92 and 100%, respectively. For ruminant PAPs processed at 133 and 137°C, the sensitivity was 100% for both 1 and 2% ruminant spikes. Overall accuracies were 96 and 99% for 1 and 2% ruminant spikes, respectively. In conclusion, the MELISA-TEK Ruminant assay showed satisfactory results, which makes it a suitable candidate method to enable safe reintroduction of non-ruminant PAPs in aqua feed.