The present study was conducted in sixty orthopedics patients who were divided into two groups, Group I with thirty patients of osteosarcoma and Group II with thirty patients with musculoskeletal pain.The present study indicated that serum folate and homocysteine levels were increased in osteosarcoma patients and decreased vitamin B12 and cholinesterase in osteosarcoma patients as compared to controls. Tumors are known to up regulate FRK, modulating the folate uptake in serum and increased homocysteine accumulation.Vitamin B12 diminishes osteoblastic activity and lowered serum vitamin B12 levels are possibly due to increased metabolic demand of the tumor. Raised homocysteine levels owing to contribution from rapidly proliferating cells resulted in decreased cholinesterase levels along with possible consumption of cholinesterase by osteoblasts in bone formation and tumorigenesis. Thus, cholinergic inhibitors, vitamin B12 supplementation, along with newer antifolate derivative and drugs targeting FRK proteins may be of value in treatment of osteosarcoma in future. The present study suggests that these parameters can serve as useful markers for diagnosis and follow up of disease.