Heparin is the most widely used anti-coagulant for the prevention of blood clots in patients undergoing certain types of surgeries including open heart surgeries and dialysis. The precise monitoring of heparin amount in patients’ blood is crucial for reducing the morbidity and mortality in surgical environments. Based upon these considerations, we have used Raman spectroscopy in conjunction with partial least squares (PLS) analysis to measure heparin concentration at clinical level which is less than 10 United States Pharmacopeia (USP) in serum. The PLS calibration model was constructed from the Raman spectra of different concentrations of heparin in serum. It showed a high coefficient of determination () between the spectral data and heparin level in serum along with a low root mean square error of prediction . It enabled the detection of extremely low concentrations of heparin in serum () as desirable in clinical environment. The proposed optical method has the potential of being implemented as the point-of-care testing procedure during surgeries, where the interest is to rapidly monitor low concentrations of heparin in patient’s blood.