AbstractFabry disease (FD) is a lysosomal storage disorder caused by variants in the GLA gene encoding α‐galactosidase A, an enzyme required for catabolism of globotriaosylceramide (Gb3). Accumulation of Gb3 in patients’ cells, tissues, and biological fluids causes clinical manifestations including ventricular hypertrophy, renal insufficiency, and strokes. This protocol describes a methodology to analyze urinary Gb3 and creatinine. Samples are diluted with an internal standard solution containing Gb3(C17:0) and creatinine‐D3, centrifuged, and directly analyzed by ultra‐high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC‐MS/MS) using an 8.7‐min method. Eight Gb3 isoforms [C16:0, C18:0, C20:0, C22:1, C22:0, C24:1, C24:0, and (C24:0)OH] are analyzed and the total is normalized to creatinine. Confirmation ions are monitored to detect potential interferences. The Gb3 limit of quantification is 0.023 µg/ml. Its interday coefficients of variation (3 concentrations measured) are ≤15.4%. This method minimizes matrix effects (≤6.5%) and prevents adsorption or precipitation of Gb3. Urine samples are stable (bias <15%) for 2 days at 21°C, 7 days at 4°C, and 4 freeze/thaw cycles, whereas prepared samples are stable for 5 days at 21°C, and 14 days at 4°C. The Gb3/creatinine age‐related upper reference limits (mean + 2 standard deviations) are 29 mg/mol creatinine (<7 years) and 14 mg/mol creatinine (≥7 years). This simple, robust protocol has been fully validated (ISO 15189) and provides a valuable tool for diagnosis and monitoring of FD patients. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Analysis of urinary globotriaosylceramide (Gb3) and creatinine by UHPLC‐MS/MSSupport Protocol 1: Preparation of the urinary quality controlsSupport Protocol 2: Preparation of the urine matrix used for the Gb3 calibration curveSupport Protocol 3: Preparation of the Gb3 calibratorsSupport Protocol 4: Preparation of the working solution containing the internal standardsSupport Protocol 5: Preparation of the creatinine calibratorsSupport Protocol 6: Preparation of the UHPLC solutions and mobile phases