Meat species adulteration is a worldwide problem, which violates food labeling laws, constitutes economic fraud, and raises ethical, religious and food safety concerns. In the US retail market, sheep is a major substituting species in other ground meats with a higher violation rate in cooked meats than in raw meats. Mixing of different species followed by grinding and/or heat-processing adds to the difficulties of discrimination of meat origin and limits the detectability of many analytical techniques such as electrophoresis and chromatography. Enzyme-linked immunosorbent assay (ELISA) is a useful tool in meat species identification. Although several commercial ELISA kits using polyclonal antibodies (PAbs) are currently available for the qualitative detection of cooked sheep meat, immunoassays using monoclonal antibodies (MAbs) would offer advantages over PAb-based immunoassay for meat speciation. This study aimed to develop a rapid MAb-based sandwich ELISA for the detection of undeclared sheep content in heat-processed meats. A pair of MAbs was previously produced using hybridoma technology by cell fusion after immunizing mice with soluble myofibril proteins extracted from heat-treated ovine muscle. MAb 7F6 (IgG1) was used as the capture antibody and MAb 6F11 (IgG2a) conjugated to biotin was used as the detection antibody. The sandwich ELISA constructed with these two MAbs displayed strong reactivity to cooked (100oC, 30 min) ovine muscle proteins. There was no observed cross-reactivity to any of the protein extracts from non-ovine meats (beef, pork, horse, deer, and poultry) and some non-flesh proteins (milk, egg albumin, and gelatin) that are commonly used as food additives in processed meat products. Only soy proteins showed a slight cross-reaction. Laboratory-adulterated cooked meat mixtures including sheep-in-pork, sheep-in-beef, and sheep-in-chicken were prepared at various adulteration levels (0% â 10%, wt/wt) in order to evaluate the sensitivity of the assay. The detection limits for cooked sheep muscle spiked in pork, beef, and chicken were 0.5%, 0.5%, and 0.25% (wt/wt), respectively. The average intra- and inter-assay coefficient of variation was 3.4% and 6.0% for lamb-in-pork, 3.5% and 4.7% for lamb-in-beef, and 3.2% and 5.9% for lamb-in-chicken, respectively. This is the first report of a MAb-based sandwich ELISA that has demonstrated utility in the authentication and/or detection of trace amounts of ovine muscle in heat-processed meat products.