Limited or absent activity of the enzyme α-galactosidase A (α-Gal A), due to mutation in the related gene on the X chromosome, leads to the development of a rare hereditary and genetic disease known as Fabry disease (FD). This pathology involves a progressive accumulation in various organs of the substrates of the enzyme e.g., globotriaosylceramide (Gb3) and its deacylated form, globotriaosylsphingosine (Lyso-Gb3), suggesting these molecules as biomarkers of Fabry disease. The present paper describes the development of an analytical strategy for the identification and quantification of Gb3 and Lyso-Gb3, in serum and blood samples by using liquid chromatography (LC) coupled to mass spectrometry in multiple reaction monitoring (MRM/MS) ion mode. The best experimental conditions were obtained by extracting the glycolipids with chloroform/methanol/H2O (2/1/0.3) and by separating them on a C4 column with a linear gradient (A: H2O with 2 mM ammonium formate. B: methanol with 1 mM ammonium formate, both acidified with 0.2% formic acid). The best transitions (a combination of precursor and fragment ions—m/z) were 786.8 m/z > 268.3 m/z for Lyso-GB3, 1137.3 m/z > 264.3 m/z for Gb3, 1039.3 m/z > 264.4 m/z for N-heptadecanoyl-ceramide trihexoside, and 843.5 m/z > 264.3 m/z for N-glycinated lyso-ceramide trihexoside, the latter being used as an internal standard. The developed method provided a reliable, fast, and effective procedure for direct measurements of GB3 and Lyso-GB3 in serum and blood for diagnosis of Fabry disease, suggesting this method as a complementary assay to the current enzymatic test. Therefore, this approach could open new insights into the clinical diagnostics of lysosomal storage disorders.