Vitamin D plays a vital role in skeletal metabolism, calcium homeostasis, and also in the functioning of the immune, cardiovascular, and reproductive systems. Vitamin D deficiency leads to rickets and osteomalacia and is also associated with breast and colorectal cancers, multiple sclerosis, dementia, rheumatoid arthritis, diabetes, parkinson’s and alzheimer’s diseases. Consequently, laboratory testing of quantitative HPLC analysis kit for 25-Hydroxyvitamin D2 and D3 in human serum & plasma samples has experienced phenomenal growth over the past several years.