A duplex PCR assay was designed for simultaneous identification of soybean and poultry in sausages. A Multi-Fast DNA Isolation Kit was used for simultaneous extraction of amplifiable soybean and poultry DNA. Primers generated specific DNA fragments of 173 and 183 bp in length for soybean and poultry, respectively. The duplex assay sensitivity was 0.35% for specific detection of poultry mixed with soybeans and other additive materials and was 0.05% for specific detection of soybeans mixed with poultry and other additive materials. The optimized duplex PCR assay was applied to 15 commercial sausages. Unintentional and intentional contaminants were analyzed using real-time PCR based Sakalar Quantification Table of DNA (SQT-DNA). Techniques developed here are suitable tools for qualitative and quantitative comparison of sausage contents.