Vitamin D is a fat-soluble vitamin that is required to perform physiological processes. It is critical for maintaining the calcium-phosphate balance, ensuring skeletal integrity, and supporting cellular functions, including proliferation, differentiation, and apoptosis. The primary sources of vitamin D in humans include dietary intake and endogenous synthesis. Additionally, vitamin D plays significant roles in immune regulation and cardiovascular health, making it a key factor in overall human health. Over the past 25 years, several 50 vitamin D metabolites have been identified. Vitamin D deficiency is prevalent globally. Analyzing the concentrations of vitamin D and its metabolites is essential for diagnosing and managing deficiencies, comprehensively understanding the role of vitamin D in health and disease. This chapter includes different types of assays, spectroscopic and chromatographic techniques each with unique advantages and limitations, ensuring precise quantification in various biological matrices. The importance of different types of chromatographic techniques, hyphenated mass spectrometers, and immunoassays are the highlights of this chapter.