In current clinical practice, a thorough understanding of vitamin D metabolism is in high demand both for patients with various diseases and for healthy individuals. Analytical techniques that provide simultaneous measurement of multiple metabolites are preferred. Herein, the development of an HPLC-DMS-MS/MS method for the quantitation of vitamin D compounds (25(OH)D3, 25(OH)D2, 1,25(OH)2D3, 3-epi-25(OH)D3, 24,25(OH)2D3, and D3) in serum is described. The selected sample preparation procedure based on the combination of liquid–liquid and solid-phase extraction, which excluded a lengthy derivatization step, was compared with other common approaches. Sensitivity was increased through the implementation of differential ion mobility separation. The proposed assay allowed us to determine the low abundant 1,25(OH)2D3with the detection limit of 10 pg/mL. The validation study showed good linearity (r2> 0.99), a wide analytical range (2.5–75 ng/mL for 25(OH)D3), and acceptable precision (<7%) for all metabolites. The recovery ranged from 71% to 93% and the matrix effect from 0.80 to 0.95 depending on the metabolite; accuracy determination was performed using DEQAS controls.