Cyanocobalamin is the synthetic form of Vitamin B12. It helps to prevent deficiency of B12 in the body. Vitamin B12 plays an important role in the formation of red blood cells and also helps in absorption of iron in the body. A cost effective and high-throughput simple preparative LC method was developed with a runtime of 35 min for the isolation of the two process impurities 50-Carboxycyanocobalamin and 32-Carboxycyanocobalamin of cyanocobalamin drug substance by preparative LC. Since the impurity level was low in the sample, after trying several days, failed to enrich the impurity level by synthetic route. A thorough study has been undertaken to develop a preparative LC method to isolate and characterize these impurities by chromatographic and spectroscopic methods. The crude sample contains low level of the impurities, about 36% and 12% were isolated by preparative HPLC with a purity greater than 95%. Keywords: Preparative LC (PREP), High performance liquid chromatography (HPLC), USP monograph spectroscopy; Isolation; Identification and Characterization