Measurement of vitamin D metabolites is no longer confined to specialist laboratories with bespoke, high end LC MS/MS instruments. For 25 hydroxyvitamin D (25(OH)D), there is now an assay format to match most needs, budgets and skill levels: high throughput automated immunoassay platforms for total 25(OH)D; commercial LC MS/MS IVDR kits and laboratory developed tests that separately quantify 25(OH)D3 and 25(OH)D2; and the recently launched fully automated Roche LC MS/MS platform. Each approach has distinct implications for analytical specificity, cross-reactivity with other vitamin D metabolites, ability to resolve the D3/D2 and C3 epimers, and alignment with standard reference materials and external quality assessment (EQA) schemes. Extending measurement to include the active and catabolic metabolites 1,25 dihydroxyvitamin D (1,25(OH)₂D) and 24,25 dihydroxyvitamin D (24,25(OH)₂D) can provide clinically relevant information on vitamin D metabolism. Vitamin D metabolite ratio (VMR) can help identify patients with impaired CYP24A1 activity, characterise vitamin D catabolic status and help explain heterogeneous responses to vitamin D supplementation. This presentation will build on these developments, summarising current analytical options for the key vitamin D metabolites and highlighting pre analytical, methodological and standardisation issues. It will also discuss how physiological and pathological states influence vitamin D metabolite profiles and VMRs, and how these measurements can be implemented in routine diagnostic work and research practice.