Newborn screening is an important public health measure of secondary prevention. With the increasing number of target conditions, there is a growing demand to optimize laboratory processes to save patient material and to work cost-effectively. Here, we report an adaption of the commercially available SpotOn Clinical Diagnostics tandem mass spectrometry test kit to detect hemoglobin fragments as well as substrate-product pairs of biotinidase and porphobilinogen synthase at once. The presence of specific peptides and the enzyme activities allows to infer to disease states, i.e., sickle cell disease, biotinidase deficiency and hereditary tyrosinemia type 1.