25-hydroxyvitamin D is mainly susceptible to hydroxylation on two positions. Hydroxylation on the 1-position by CYP27B1 yields the hormonally active 1,25-dihydroxyvitamin D, while hydroxylation on the 24-position yields the hormonally inactive 24,25-dihydroxyvitamin D. The amount of the hormonally inactive metabolite 24,25(OH)2D is dependent on the amount of its predecessor 25(OH)D and the activity of the enzyme responsible for its production. The 25-hydroxyvitamin D/24,25-dihydroxyvitamin D ratio is thus indicative for the efficiency of this catabolic clearance by CYP24A1. As such, it can identify the rare genetic disorder idiopathic infantile hypercalcemia. The disease is caused by an inactivating mutation in Cyp24A1, resulting in an absence of 24,25-dihydroxyvitamin D and an overproduction of 1,25-dihydroxyvitamin D. We here describe validation parameters of a newly developed LC-MS/MS method to determine 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D simultaneously in blood and determination of reference values for 24,25-dihydroxyvitamin D and the ratio, obtained from a well-described diverse, and healthy cohort (N=92, 50% female, aged 21-71). For 24,25-dihydroxyvitamin D reference values are 0.5 to 11 nmol/L. For the 25-hydroxyvitamin D/24,25-dihydroxyvitamin D ratio reference values are 8-27. In conclusion, we have established reference values for 24,25-dihydroxyvitamin D using a newly developed accurate LC-MS/MS method.