The interest in early detection strategies for lysosomal storage disorders (LSDs) in newborns and high-risk population has increased in the last years due to the availability of novel treatment strategies coupled with the development of diagnostic techniques. We report the development of a short-incubation mass spectrometry-based protocol that allows the detection of Gaucher, Niemann-Pick A/B, Pompe, Fabry and mucopolysaccharidosis type I disease within 4h including sample preparation from dried blood spots. Optimized sample handling without the need of time-consuming offline preparations, such as liquid-liquid and solid-phase extraction, allows the simultaneous quantification of five lysosomal enzyme activities using a cassette of substrates and deuterated internal standards. Applying incubation times of 3h revealed in intra-day CV% values ranging from 4% to 11% for all five enzyme activities, respectively. In a first clinical evaluation, we tested 825 unaffected newborns and 16 patients with LSDs using a multiplexed, turbulent flow chromatography-ultra high performance liquid chromatography-tandem mass spectrometer assay. All affected patients were identified accurately and could be differentiated from non-affected newborns. In comparison to previously published two-day assays, which included an overnight incubation, this protocol enabled the detection of lysosomal enzyme activities from sample to first result within half a day.