Vitamin D metabolism is actively modulated in adipose tissue during obesity. To better investigate this process, we develop a specific LC-HRMS/MS method that can simultaneously quantify three vitamin D metabolites, i.e., cholecalciferol, 25-hydroxyvitamin D₃ (25(OH)D₃), and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in a complex matrix, such as mouse adipose tissue and plasma. The method uses pretreatment with liquid–liquid or solid–phase extraction followed by derivatization using Amplifex^® reagents to improve metabolite stability and ionization efficiency. Here, the method is optimized by co-eluting stable isotope-labelled internal standards to calibrate each analogue and to spike biological samples. Intra-day and inter-day relative standard deviations were 0.8–6.0% and 2.0–14.4%, respectively for the three derivatized metabolites. The limits of quantification (LoQ) achieved with Amplifex^® derivatization were 0.02 ng/mL, 0.19 ng/mL, and 0.78 ng/mL for 1,25(OH)₂D₃, 25(OH)D₃ and cholecalciferol, respectively. Now, for the first time, 1,25(OH)₂D₃ can be co-quantified with cholecalciferol and 25(OH)D₃ in mouse adipose tissue. This validated method is successfully applied to study the impact of obesity on vitamin D status in mice.