This chapter describes a high-pressure liquid chromatography (HPLC) method for measuring aflatoxin B1 and its metabolites isolated from rat hepatocytes. AFB1, AFQ1, AFM1, and AFP1 concentrations are determined using reverse-phase HPLC. The isocratic mobile phase is 18% dimethylformamide in 0.01% phosphoric acid at a flow rate of 2.0 mL/min at 69°C, with ultraviolet (UV) detection at 360 nm. AFB-GSH conjugate levels are also measured. An isocratic mobile phase consisting of 69.05% H2O, 30% methanol, and 0.05% acetic acid is used and the sample is eluted at ambient temperature with a flow rate of 1.0 mL/min and UV detection at 365 nm.