This study was conducted to supplement limit of previous study, The objectives of this study were to select optimal conditions of high performance liquid chromatography(HPLC) operation for detecting urinary 2-thiothiazolicline-4-carboxylic acid(TTCA) and thiocarbamide simultaneously, and to evaluate recovery rates for various liquid-liquid extration method of these metabolites, The results are as follows : 1. The urinary TTCA and thiocarbamide were separate sharply when flow rate is $0.7m{\ell}/min$, using a series $C_8$ and C18 column, 50 mM KH2PO4 : acetonitrile (93.5 : 6.5) and pH 3.5 as a mobile phase. The retention time was TTCA, $12.07{\pm}0.11$(mean±SD, n=06), thiocarbamide, $7.85{\pm}0.01$ (mean±SD, n=6), respectively. The calibration curve for TTCA and thiocarbamide was linear within the range 0.05 to $30{\mu}g/m{\ell}$. 2. By the liquid-liquid extration, butanol extration with $(NH_4)_2$ as a salting-out reagent was used as a simultaneous extration method for these metabolites in acid state, and recovery rates of this method are urinary TTCA, $49.6{\pm}17.7$ (mean±SD, n=16), thiocarbamide, $43,9{\pm}5.50$ (mean±SD, n=16), respectively 3. The precision(pooled coefficients of variation for 4 concentration) of the urinary thiocarbamide analysis was 0.03754 by butanol liquid-liquid extraction with $(NH_4)_2$ as a salting-out reagent, and TTCA was 0.04082 by ethyl acetate liquid-liquid extration with $(NH_4)_2$ as a salting out reagent The above results show that the butanol liquid-liquid extraction with $(NH_4)_2$ as a salting-out reagent in acid state, and using a series $C_8$ and C18 column, 50 mM KH2PO4 : acetonitrile (93.5 : 6.5) and pH 3.5 as a mobile phase are suitable for the analysis of urinary TTCA and thiocarbamide simultaneously. The detection limit of TTCA and thiocarbamide was about $0.17{\mu}g/m{\ell}$, $0.07{\mu}g/m{\ell}$.