A fast method for the measurement of metabolites of pyrene in urine was improved by HPLC with fluorescence detector using “heart-cut” technique. This method can quantify the total amount of pyrene metabolites corresponding to glucuronic acid and sulfate conjugates as well as free 1-OH-Py. The hydrolyzed biological fluid was directly injected into the chromatographic system, via a column-switching system. Pre-treatment and analysis were performed within 0.5 and 9 min, respectively. Enzymatic hydrolysis has been optimized to not exceed 2 h. The best response function, in the 0.2–10 ng/mL range, is the linear regression, bringing simplicity, good accuracy, and low LOQ. The intra- and interday precision values were inferior to 1 and 2%, respectively. The proposed method provided a simple, convenient, and practical procedure to determine the level of the main urinary pyrene metabolites in biological samples.