ABSTRACT Rutin is a polyphenolic naturally occurring compound potentially having a huge impact in human benefit which includes wound healing, cardiovascular, carcinoma therapeutic activities as well as have nutraceutical benefits. The objective of study was to establish a precise, accurate, sensitive and reliable method for estimation at low concentrations of rutin in biological fluids, using spectrofluorimetric method.A standard curve of rutin was prepared in methanol and serum in the concentration range 1-10 ng/ml. The method was validated in terms of linearity, accuracy and precision.The regression data for rutin in methanol and serum observed to have a good linear relationship with R 2 > 0.99 over the concentration range of 1-10 ng/ml. This method gave adequate linearity, precision, accuracy and recovery. It is also concluded that method is less time consumption as compared with HPLC, which requires lengthier chromatographic separation