Objective To evaluate the performance of a statewide full-population pilot study in Missouri on newborn blood spots for screening of lysosomal storage disorders (LSDs) using digital microfluidics. Study design A full-population pilot study using a multiplexed fluorometric enzymatic assay to detect Pompe disease, Fabry disease, Gaucher disease, and mucopolysaccharidosis type I (MPS I) in the Missouri newborn population is ongoing. Provisional cutoff values were determined during a prepilot study. All newborn dried blood spots received at the Missouri State Public Health Laboratory for routine newborn screening were screened for the 4 LSDs during the pilot study. Newborns determined to be screen-positive were referred for confirmatory testing. Results The study commenced on January 11, 2013; during the first 6 months, 43 701 specimens were screened, and 27 newborns with a confirmed diagnosis of an LSD genotype (8 with Pompe disease, 1 with Gaucher disease, 15 with Fabry disease, and 3 with MPS I) were identified. These numbers correspond to detection rates of 1:5463 for Pompe disease, 1:43 701 for Gaucher disease, 1:2913 for Fabry disease, and 1:14 567 for MPS I. The positive predictive values were 47% for Pompe disease with 1 lost to follow-up, 10% for Gaucher disease, 58% for Fabry disease with 2 lost to follow-up, and 11% for MPS I with 4 pending. Conclusion The first 6 months of the Missouri LSD pilot study provided the opportunity to validate the effectiveness of the digital microfluidic screening method, refine the cutoffs for detection of these LSDs, and test the entire system of infant referral, follow-up, confirmation, treatment, and screening program communication.