In this study, surface-enhanced Raman spectroscopy (SERS) based quantification method for the total protein using o-phthalaldehyde is reported for the first time. For this purpose, o-phthalaldehyde was chosen to form a complex with protein, and SERS signal was observed in the presence of gold nanoparticles. A calibration curve was obtained by plotting the intensity of the SERS signal at 727cm(-1)versus the concentration of protein standard (bovine serum albumin, BSA). Thus, the correlation was found to be linear within the range of 0.054-0.72mg/ml of BSA, and the limit of detection was determined to be 0.08mg/ml. All tests were carried out using a portable Raman instrument with an analysis time of 5min. In addition, the ability of the developed method to quantify total protein in milk samples was investigated, and the obtained results were compared to the conventional ultraviolet methods. Copyright (c) 2017 John Wiley & Sons, Ltd.