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Background: Niemann Pick disease (NP) is a rare, lysosomal storage disorder due to deficiency of the intra-lysosomal enzyme acid sphingomyelinase (ASM) resulting in intracellular accumulation of sphingomyelin. We evaluated a tandem mass spectrometry (MS/MS) method to analyze ASM activity in dried blood spots (DBS) that may be suitable for laboratory diagnosis of NP including high throughput screening of at-risk populations and potentially for newborn screening., Methods: ASM activity was measured in 3.2 mm punches from DBS. The eluate was incubated with the ASM substrate (N-Hexanoyl-D-erythro-sphingosylphosphorylcholine [C6-sphingomyelin (C(29)H(59)N(2)O(6)P)]) and an internal standard (N-butyroyl-D-erythro-sphingosine [C4-ceramide (C(22)H(43)NO(3))]). ASM product and IS were analyzed using MS/MS in multiple reaction monitoring mode for transitions m/z 370.6>264.3 (ASM internal standard) and m/z 398.6>264.3 (ASM product)., Results: ASM activities were stable for up to 2 months at or below 4℃. Position of the punch in the DBS and/or hematocrit of the DBS had a limited effect on ASM activities. Both intra- and inter-assay variability were below 10%. There was no carry-over. The median ASM activity in 2,085 newborn infants was 9.5 µmol/h/L (mean 10.6) with a SD of 5.06 µmol/h/L. Six of 2,085 (0.3%) infants were found to have ASM activities below the cut-off of 2.5 µmol/h/L. ASM activities were below the cut-off level in all 10 previously diagnosed cases with NP (range: 0.16 to 2.08 µmol/h/L)., Conclusions: This MS/MS method for the measurement of ASM activity in DBS is robust and suitable for laboratory diagnosis of NP.