Blood Brain Barrier (BBB) permeability is frequently compromised in the course of diseases affecting the central nervous system (CNS). Sucrose is a low molecular weight, hydrophilic marker with slow permeability at the naive BBB and therefore one of the widely used indicators of barrier integrity. Our laboratory recently developed a highly sensitive UPLC-MS/MS method for stable isotope labeled [¹³C₁₂]sucrose in biological matrices. Correction of total brain concentration for contribution of intravascular space is required in such experiments in order to accurately measure BBB permeability, and it is often accomplished by vascular perfusion with buffer solutions prior to brain sampling. The purpose of the present study was to develop a UPLC-MS/MS method, which allows simultaneous analysis of two different stable isotope labeled sucrose variants, one of which can be utilized as a vascular marker. The first analyte, [¹³C₁₂]sucrose, serves to quantify brain uptake clearance as a measure of BBB permeability, while the second analyte, [¹³C₆]sucrose, is administered just before termination of the animal experiment and is considered as the vascular marker. [²H₂]sucrose is used as the internal standard for both ¹³C labeled compounds. Because the majority of recent studies on CNS diseases employ mice, another objective was to validate the new technique in this species. The UPLC-MS/MS method was linear (r² ≥ 0.99) in the tested concentration ranges, from 10 to 1000 ng/mL for both analytes in plasma, from 2 to 400 ng/g [¹³C₁₂]sucrose in brain and from 10 to 400 ng/g [¹³C₆]sucrose in brain. It was also validated in terms of acceptable intra and inter run accuracy and precision values (n = 5). The dual analyte technique was applied in a study in mice. One group received intravenous bolus injections of 10 mg/kg [¹³C₁₂]sucrose at time 0, and 10 mg/kg [¹³C₆]sucrose at 14.5 min, and subsequent terminal blood and brain sampling was performed at 15 min. For comparison, another group received an intravenous bolus dose of 10 mg/kg [¹³C₁₂]sucrose and was submitted to transcardiac perfusion with buffer after 15 min. We demonstrate that the two alternative techniques to correct for intravascular content deliver equivalent values for brain concentration and brain uptake clearance.