BACKGROUND: Where conventional blood sampling is challenging, dried blood spots (DBS) provide a practical sample alternative for measuring vitamin D levels. Our study aimed to develop and evaluate a clinical pathology service-based assay suitable for measuring vitamin D in batches of DBS samples collected remote to the testing site. METHODS: A high throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with derivatisation was developed to measure 25-hydroxyvitamin D metabolites (25OHD3, 25OHD2 and 3-epi-25OHD3) in DBS samples. The assay was validated using paired DBS and plasma samples from 37 healthy adults. RESULTS: The assay reproducibly (<11.5% coefficient of variation) quantified 25OHD3 (range 1-300nmol/L), 25OHD2 (range 2-300nmol/L) and 3-epi-25OHD3 (range 1-200nmol/L) in DBS samples. The 25OHD3 metabolite was detected in all DBS samples, 3-epi-25OHD3 in six plasma (range 2.1-6.3 nmol/L) and paired DBS samples, and 25OHD2 was not detected. Concentrations of 25OHD3 were highly correlated between paired samples: capillary DBS and venous plasma (r=0.92), venous DBS and venous plasma (r=0.93), and capillary DBS and venous DBS (r=0.97). Ordinary least squares regression was used to characterise (β=0.81) and correct the systematic bias in DBS data (compared to paired plasma). Thereafter, Bland-Altman analysis demonstrated robust agreement between sample-methods. CONCLUSION: This simple and rapid DBS-based LC-MS/MS assay accurately quantified serum vitamin D metabolites using a paired-sample 'bridging strategy' to correct for the inherent sample-method bias.