Vitamin D is an essential micronutrient for bone health and the general cellular functions of the body. Its insufficiency/deficiency leads to the pathophysiology of disorders like diabetes, cancer, autoimmune, neurodegenerative, and cardiovascular diseases. Clinical interest in Vitamin D metabolites and their role in various medical disorders have contributed to an increase in laboratory demands for vitamin D measurements. For clinical and research laboratories worldwide, analysis of vitamin D and associated metabolites is a significant problem. The best way for determining vitamin D levels is constantly being debated. Various methods such as immunoassays and chromatographic techniques are available for determining vitamin D levels. Additionally, biosensors have recently been considered promising options for routine vitamin D analysis. The existing methods and other developments in the measurement of vitamin D metabolites and associated analytical challenges are discussed in this review.