1,25-dihydroxyvitamin D [1,25(OH)2D], which is the major biologically active form of vitamin D, is a hormone that helps regulate the body’s calcium and phosphate metabolism. Cholecalciferol (vitamin D3) and ergocalciferols (vitamin D2) are two different categories or families of vitamin D. To determine the nutritional status of vitamin D in the blood, estimation of 25(OH)D in the blood is considered as an ideal way, while the measurement of 1,25(OH)2D is used in the diagnosis of vitamin D-dependent rickets. Measurement of 25(OH)D and 1,25(OH)2D involves three steps, extraction, purification, and quantification. Techniques available to measure 25(OH)D include competitive protein binding assay, competitive and noncompetitive immunoassay, ultraviolet absorption, and mass spectrometry. For the estimation of 1,25(OH)2D Radioimmunoassay or RIA is commonly used. 25(OH)D in serum is considered normal between 10 and 65 ng/mL and 1,25(OH)2D is considered normal between 15 and 60 pg/mL. These are the most commonly used reference ranges, but the levels depend on the method used, ethnicity, sun exposure and other factors like pregnancy. Screening for vitamin D deficiency is suggested in high-risk individuals which include breastfed infants, strict vegetarians, darker-skinned individuals, and the elderly. Supplementation with 50,000 IU/week for 12 weeks or 50,000 IU 3 times/week for 6 weeks is recommended in patients with 25(OH)D levels less than 20 ng/mL.