Objective：To optimize and establish the experimental methods for the determination of 2,4-dihydroxybenzophenone (BP-1) in mouse brain. Methods: BP-1 was determined by high performance liquid chromatography (HPLC) and separated by Waters Symmetry^® C18 (4.6 mm×250 mm, 5 μm) using isocratic elution, and the sample preparation conditions were optimized by orthogonal experiment design. The mobile phase was methanolwater (volume ratio 3∶1) containing 3% (volume fraction) acetic acid (pH 3.40) at a flow rate of 1.0 mL/min, and ultraviolet (UV) detection wavelength was set at 290 nm. Retention time was used for qualitative analysis and internal standard method for quantitative analysis. Results: Under the optimized experimental conditions, the calibration curve was linear with a correlation coefficient of 0.999 8 over the concentration range of 0.2-10.0 mg/L. The recoveries of BP-1 were between 96.8% and 104.5%. The intra-day and inter-day precision of BP-1 were 3.5%-5.7% and 4.5%-6.4%, respectively. The extraction recoveries of BP-1 at three concentrations (0.5, 2.0, 8.0 mg/L) in the mouse brain were 90.5%, 89.5%, and 97.7%, and the matrix effect of BP-1 at these three concentrations were 102.9%, 102.7%, and 90.9%, respectively. Conclusion:The method is simple, accurate, and suitable for determination of the contents of BP-1 in mouse brain.