Vitamins play an important role in many processes in the human organism. The detection of insufficient supply of vitamins is therefore of particular importance to avoid significant effects for human health. An increasing number of tests is only possible with suitable automated procedures. For the determination of vitamin D3 and vitamin D2 in serum samples, three methods were automated and compared with regard to their performance. All three methods enable reliable detection of 25(OH)D2 and 25(OH)D3 in serum in the ng/ml range.