Food fortification is an effective approach to improve vitamin D (VD) concentrations in foods. Eggs are a useful food vehicle for enrichment with VD via its hydroxylated metabolite, 25-hydroxyvitamin D (25-D3), in hen feed. This study determined the impact of time of lay, storage conditions (ambient and refrigeration) and common cooking methods (boiling, frying, scrambling, poaching and microwaving) on the vitamin D metabolite concentration of eggs enriched with 25-D3. Processed samples were freeze-dried and analysed for D3and 25-D3using an HPLC-MS(/MS) method. The results indicated that storage and cooking practices influence VD metabolites, with 25-D3showing true retention of 72–111% and concentrations of 0.67–0.96 µg/100 g of whole egg. Vitamin D3showed true retention of 50–152% and concentrations of 0.11–0.61 µg/100 g of whole egg. Depending on the storage and method of cooking applied, the calculated total VD activity of enriched eggs ranged from 3.45 to 5.43 µg/100 g of whole egg and was 22–132% higher in comparison to standardised VD content for non-enriched British eggs. The study suggests that 25-D3is a stable metabolite in eggs following storage and cooking, and that 25-D3-enriched eggs may serve as a potent dietary source of VD.