Deficiencies in any of the 50 degradative enzymes found in lysosomes results in the accumulation of undegraded material and subsequently cellular dysfunction. Early identification of deficiencies before irreversible organ and tissue damages occur leads to better clinical outcomes. In the method which follows, lysosomal α-glucosidase, α-galactosidase, β-glucocerebrosidase, acid sphingomyelinase, and galactocerebrosidase are extracted from dried blood spots and incubated individually with an enzyme-specific cocktail containing the corresponding substrate and internal standard. Each enzyme cocktail is prepared using commercially available mixture of substrate and internal standard at the predetermined optimized molar ratio. After incubation, the enzymatic reactions are quenched using an ethyl acetate/methanol solution and all five enzyme solutions are combined. The mixtures of the reaction products are prepared using liquid–liquid and solid-phase extractions and quantified simultaneously using selected ion monitoring on LC-MS-MS system.