A high performance liquid chromatographic(HPLC) method was developed for the direct and simultaneous determination of cystine,tyrosine and histidine in hair of rat.The chromatographic separation and detection conditions were optimized,and the samples were analyzed after acid hydrolysis.The determination was performed on a Titank C18 column(4.6 mm×250 mm,5 μm) with acetonitrile-10 mmol/L diammonium hydrogen phosphate buffer(containing 10 mmol/L sodium 1-octanesulfonate and obtaining pH 2.0 by phosphoric acid) as mobile phases by gradient elution at a flow rate of 1.0 mL/min.The detection wavelength was set at 205 nm,and the injection volume was 100 μL.The column temperature was set at 8 ℃.Under the optimal conditions,cystine,tyrosine and histidine in hair of rat were effectively separated．Good linearities for the analytes were obtained in their respective ranges,with correlation coefficients(r) larger than 0.999.The limits of detection(S/N=3) were in the range of 49- 442 μg/L and the limits of quantitation(S/N=10) were 148-884 μg/L.The spiked recoveries were in the range of 97.8%-102% with relative standard deviations of 0.1%-1.6%.This method is simple,accurate,reliable and reproducible,and could be applied in the determination of cystine,tyrosine and histidine in hair of rat.