Lysosomal storage disorders (LSDs) are a very heterogeneous group of hereditary disorders. The diagnostic process usually involves complex sampling, processing, testing, and validation procedures, performed by specialized laboratories only, which causes great limitations in reaching a diagnosis for patients affected by these diseases.There are few studies about LSDs in Colombia. The diagnostic limitations often make medical practitioners disregard the possibility of these disorders while diagnosing their patients. The current study documents the results of a 7-year screening in high-risk patients, aimed to detect LSDs using dried blood spots (DBS) collected on filter paper, with a micromethodology that facilitates diagnosis even with a large number of samples.The activities of α-galactosidase A, α glucosidase, α-L-iduronidase, arylsulfatase B, β-galactosidase, β-glucosidase, total hexosaminidase, iduronate sulfatase, and chitotriosidase were analyzed in high-risk patients for lysosomal disease. The catalytic activity was evaluated with fluorometric micromethods using artificial substrates marked with 4-methylumbelliferone.The reference values for a control population were established for the enzymes listed above, and 242 patients were found to have an enzyme deficiency, guiding to the following diagnoses: Fabry disease (n = 31), Pompe disease (n = 16), Hurler Syndrome (n = 15), Maroteaux-Lamy Syndrome (n = 34), GM1 Gangliosidosis (n = 10), Morquio B (n = 1), Gaucher disease (n = 101), Sandhoff disease (n = 1), Mucolipidosis (n = 2), and Hunter Syndrome (n = 31). In conclusion, this protocol provides a comprehensive diagnostic approach which could be carried out in Colombia and made it available to medical services spread around the country, enabling the identification of a large number of patients affected by LSDs, which could potentially benefit from the therapeutic tools already available for many of these diseases.