A simple procedure for separation of ribose-5-phosphate, deoxyribose-1-phosphate and ribose-1-phosphate is based on high performance liquid chromatography using reversed phase 4 × 300 mm “μ Bondapak/NH2” column. The column is equilibrated with 0.13 M borate buffer (pH 7.5) followed by gradient elution of ribose-5-phosphate, deoxyribose-1-phosphate and ribose-1-phosphate using water, 0.05 M borate buffer containing 0.1 M MgCl2(pH 9.6) and 0.05 M sodium acetate-acetic acid buffer containing 0.1 M MgCl2(pH 5.0) as eluants respectively. Eluates of borate complex “μ Bondapak/NH2” column are brought to pH 9.6 by the addition of 1 N KOH and enzymatically hydrolysed with alkaline phosphatase (EC 3.1.3.1) to release the free pentoses. The free pentoses are mixed with a reagent solution prepared from aqueous solution of 2% cyanoacetamide and 0.6 M borate buffer (pH 9.6), and the mixture is boiled for 10 minutes and the absorbance of the product is measured at 276 nm using a spectrophotometer.