Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A (α-GAL A) deficiency. This enzyme contributes to the cellular recycling of glycosphingolipids such as galabiosylceramide (Ga2), globotriaosylceramide (Gb3), and globotriaosylsphingosine (lyso-Gb3) by hydrolyzing the terminal α-galactosyl moiety. Urine and plasma α-GAL A substrates are currently analyzed as biomarkers for the detection, monitoring, and follow-up of Fabry disease patients. The sensitivity of the analysis of Ga2 is decreased by the co-analysis of its structural isomer, lactosylceramide (LacCer), which is not an α-GAL A substrate. A normal-phase ultraperformance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) methodology, allowing the baseline separation of 12 Ga2 isoforms/analogues from their lactosylceramide counterparts, was developed and validated in urine. The method was multiplexed with the analysis of 12 Gb3 isoforms/analogues having the same fatty acid moieties as those of Ga2 for comparison, and with creatinine for sample normalization. Urine samples were studied from 34 untreated and 33 Fabry males treated by enzyme replacement therapy (ERT) and 54 untreated and 19 ERT-treated Fabry females, along with 34 male and 25 female healthy controls. The chromatographic separation of Ga2 from LacCer increased the sensitivity of analysis, especially in women. One untreated Fabry female and two treated Fabry females presented abnormal levels of Ga2 but normal levels of Gb3, supporting the importance of analyzing Ga2, in addition to Gb3. Our results show that urine LacCer levels from females were significantly higher than those from males. Moreover, LacCer levels were not affected by Fabry disease for both males and females.