Analytical methodology to detect ricin and Shiga toxins (Stx) in food matrices is important for food safety and biosecurity. Monoclonal antibodies (mAbs) that bind each toxin were used for capture in sandwich enzyme-linked immunosorbent assay, and IgY polyclonal antibodies were prepared as detection antibodies. The ricin assay systems, using colorimetric or chemiluminescent substrates, detected native, but not heat-denatured ricin. The lower limit of detection (LOD) was 0.13 ng mL−1 in milk and 0.8 ng g−1 in ground beef. The Stx2 assay systems detected native Stx2, but not heat-denatured Stx2 or Stx1. The LOD was 0.13 ng mL−1 in milk and 0.7 ng g−1 in ground beef. Using a standard 96-well-plate format, the assays can detect less than 1 × 10−4 of an estimated lethal oral dose of either toxin in a serving of milk. The IgY detection antibodies for ricin were more heat-stable than mouse polyclonal anti-ricin at 65°C.