A monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the sensitive detection of porcine skeletal muscle in raw and heat-processed meat and feed products. Heat treatment of meat samples up to 132 °C for 2 h did not affect the assay performance. The assay uses a pair of monoclonal antibodies (MAbs 8F10 and 5H9) specific to skeletal muscle troponin I (TnI). MAb 8F10, reacting to mammalian TnI, is the capture antibody and the biotin-conjugated MAb 5H9, specific to porcine TnI, the detection antibody. The sandwich ELISA is able to detect 0.05% (w/w) of laboratory-adulterated pork in chicken, 0.1% (w/w) pork in beef mixtures, 0.05% (w/w) pork meal in soy-based feed, and 1% commercial meat and bone meal (MBM), containing an unknown amount of pork, in soy-based feed. This new assay provides a rapid and reliable means to detect the contamination of meat and feed products with trace amounts of porcine muscle tissue to ensure product quality and safety.