Concentrations of synthetic antioxidants butylated hydroxyanisole, butylated hydroxytoluene, and tert-butyl hydroquinone were quantified using a high-performance liquid chromatograph with spectrofluorometric detector. The antioxidants were separated and eluted on a reversed-phase column by gradient of a mixture of H2O/acetonitrile/acetic acid (66.5: 28.5:5, by vol) and a mixture of acetonitrile/acetic acid (95:5, vol/vol). The eluants were monitored at emission and excitation wavelengths of 310 and 280 nm, respectively. Calibration curves obtained using peak areas against concentration showed high coefficients of multiple determination (R2 > 0.99) for all antioxidants. Known concentrations of added antioxidant standards were recoverable within 98-99% from oils and over 93% from mouse blood. This method requires minimum sample extraction and purification before analysis and provides a relatively high percentage recovery. The method has been applied successfully for the measurement of antioxidant concentrations in oils, dried foods, and biological fluids.