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• As alternative for blood obtained by venipuncture dried blood spot can be considered for measuring 25-hydroxyvitamin D 3. • There is a good agreement between the measurement of 25-hydroxyvitamin D 3 obtained with dried blood spot and serum. • Dried blood spot can exclude vitamin D deficiency, but it is less suitable for individualized vitamin D status assessment. Blood for determining 25-hydroxyvitamin D 3 [25(OH)D 3 is usually obtained through venipuncture although, as an alternative for serum, dried blood spot (DBS) can be considered. The aim of this proof-of-concept study was to investigate levels of agreement between measurements of 25(OH)D 3 obtained with DBS compared with serum. 301 Chinese participants were included who completed 25(OH)D 3 measurement from DBS and from simultaneously collected blood samples obtained by venipuncture. Measurements of both DBS and serum 25(OH)D 3 were performed using liquid chromatography followed by tandem mass spectrometry. Agreement between the two methods was assessed with Passing and Bablok regression analysis and Bland-Altman plot. Measurements showed a good correlation (Pearson's correlation coefficient r = 0.929, P < 0.001) between the two methods. After recalculating for a 13% difference, a regression equation of DBS 25(OH)D 3 = -1.91 + 1.00 serum 25(OH)D 3 was found in Passing and Bablok regression analysis. Bland-Altman analysis showed a fixed bias of 1.7 nmol/L; upper and lower limit of agreement was 24.1 nmol/L and -20.7 nmol/L, respectively. Sensitivity of recalculated DBS for 25(OH)D 3 concentrations <30 and <50 nmol/L was 87.8% and 91.1%, respectively, and specificity was 89.2% and 83.1%, respectively. In conclusion, a good agreement was found between the measurement of 25(OH)D 3 obtained with DBS compared with serum. DBS may possibly be used in a future screening program, but it is less suitable for individualized vitamin D status assessment. [ABSTRACT FROM AUTHOR]