A method was developed and validated for low-level detection of glucose. The method involves quantitation of glucose though derivitisation with PABA and HPLC-DAD analysis. A selective and novel method has been optimised for evaluation of blood glucose levels in blood Serum and saliva biological matrices by R.P-H.P.L.C. The principle analyte was eluted with the conditions of mobile phase having the 50m.M Sodium acetate: Acetonitrile (60:40%, v./v.) using the Phenomax-C.18 (250 x 4.6 mm, 3.5m.) analytical column with the 1.0 ml/min flow rate and 10µl sample volume at 254 nm in a photodiode array detector. The retention times of was 3.4 min within the total run time of 05 min. The curve indicates the correlation coefficient (r2) was superior by having the value 0.998 with a linear range of 40 µg/ml- 600.0 µg/ml. Based on the results obtained in the validation, the developed method was susceptible, accurate, linear and economical. Due to the short time of the chromatographic program, more samples can be analysed within a short period. Data obtained were subjected to statistical analysis using t-test. Highly significant P-value (P<0.05) was obtained between the serum blood glucose and salivary glucose level. A distinct difference was observed in the salivary glucose between the control and diabetic group. The method was met all the predefined acceptance criteria. Diabetes mellitus is a globally widespread disease. As the salivary collection is painless and non-invasive, in this study, an attempt has been made to diagnose diabetes mellitus by estimating the salivary glucose level in comparison with serum blood glucose level. Hence the developed method can be used as an index of diabetes mellitus.