We developed a novel and straightforward derivatization method for the determination of lactate by reversed phase high-performance liquid chromatography (RP-HPLC) with fluorescence and UV detection in biological matrices as urine and saliva. The derivatization of lactate was achieved employing 9-chloromethyl anthracene (9-CMA) as fluorescence reagent, which has never been previously used to obtain a lactate derivative. Lactate reacts with 9-CMA with high selectivity in a very short time, without requiring extraction procedures from the aqueous solution, and the reaction reaches 70% completion in 30min. The ester derivative obtained can be easily determined by RP-HPLC with fluorescence detection at 410nm (λex=365nm) and UV detection at 365nm. The method was also optimized in order to allow for the simultaneous determination of lactate and creatinine for the application to urine samples. The lactate calibration curve was linear in the investigated range 2×10(-4)-3×10(-2)mM and the limit of detection, calculated as three times the standard deviation of the blank divided by the calibration curve slope, was 50nM for both fluorescence and UV detection. Intra-day and inter-day repeatability were lower than 5% and 6%, respectively. The method proposed was successfully applied to the analysis of urine and saliva samples.