A reversed-phase high-performance liquid chromatography method based on modified pre-column derivatization with phenylisothiocyanate was applied to quantify free amino acids in a protein hydrolysate extracted from waste activated sludge. The protein solution was hydrolyzed with neutral and alkaline proteases. The analytical parameters including linearity (r2≥0.99) , precision (2.0-3.0%, RSD), accuracy (97.3-103.6% recovery), limits of detection (0.002-1.71 pM) and quantification range (0.065-5.70 pM) were determined.