► A rapid and sensitive method for simultaneous analysis of Vit D and 25OH-Vit D in meats was developed. ► Samples were saponified, underwent solid phase extraction and were analysed by LC–IT-MS. ► LOD and LOQ for Vit D and 25OH-Vit D were 0.03 and 0.05μg/100g respectively. ► Recoveries using internal/surrogate standard correction ranged from 80 to 100% for all vitamers. ► Measurement uncertainty ranged from 6 to 15% for all vitamers in this method. This paper reports a method for the rapid, sensitive and simultaneous analysis of vitamin D (Vit D) and 25-hydroxyvitamin D (25OH-Vit D) in meats. Samples were saponified and underwent solid phase extraction with analysis by normal phase liquid chromatography (LC) with ion trap mass spectroscopy (IT-MS), using positive polarity atmospheric pressure chemical ionisation (APCI). Limits of detection (LOD) and quantification (LOQ) for Vit D and 25OH-Vit D were 0.03 and 0.05μg/100g respectively. Deuterium labelled Vit D and 25OH-Vit D internal standards were added as surrogates prior to saponification, correcting for extraction inefficiencies and potential MS matrix enhancement or suppression effects. Recoveries using internal/surrogate standard correction ranged from 80% to 100% for all vitamers. Measurement uncertainty ranged from 6% to 15% for all vitamers in this method. This process required only 7.5g of sample per extraction and a batch of 28 extractions could be completed in six hours.