A radioimmunoassaay (RIA) procedure to measure aflatoxin B₁ (AfB₁) in agricultural commodities was developed. AfB₁ oxime derivative was synthesized, characterized, and used for preparation of ¹²⁵I-labeled AfB₁. Antiaflatoxin B₁ serum was raised in-house using AfB₁- bovine serum albumin conjugate as immunogen. The assay system was optimized in the range of 0.2-5 ng/mL, using a liquid phase (PEG) as well as a solid phase (coated polystyrene beads) separation system. Interassay and intra-assay variations, recovery, and parallelism studies validated the assay. AfB₁ analysis was carried out in nearly 130 samples of different agricultural commodities. The correlation coefficient was determined using commercial ELISA and in-house-developed RIA methods.