A rapid, sensitive, and selective method for the quantification of vitamin D3 (cholecalciferol) in solid and liquid food, feed, and tablets based on HPLC/MS/MS has been developed and validated. The sample preparation procedure consists of a quick and robust alkaline saponification and liquid-liquid extraction, followed by direct injection of the organic extract into the HPLC/MS/MS system for analysis without any further concentration, reconstitution, or prepurification steps. The reduction in sample preparation time was achieved by applying a heart-cutting, two-dimensional chromatography technique prior to positive electrospray ionization selected reaction monitoring MS analysis. Total vitamin D3 (sum of previtamin D3 and vitamin D3) was quantified using an isotopically labeled internal standard. The ionization efficiency of previtamin D3 and vitamin D3 in the positive electrospray ionization mode was found to be very similar. The validation experiments included four feed matrixes, three types of tablets, and 12 food matrixes. The obtained recoveries were between 96.1 and 105.3%, and intermediate precision ranged from 1.32 to 15.6% RSD, with HorRat values between 0.07 and 0.65. For all samples, extraction efficiencies were above 95.8%. Analysis of two certified reference materials (SRM 1849 and BCR-122) gave accuracies of 102.4 and 99.8%, respectively.