Laser ablation (LA) is a powerful analytical technique for solid microsampling. Its coupling with ICPMS has been shown to offer good precision and accuracy for the elemental analysis of glass fragments. Fractionation in LA poses one of the major challenges to using this technique for in situ trace elemental profiling of glass evidence. The aim of this work was to study the effect of elemental fractionation on the forensic application of elemental analysis of glass samples by LA-ICPMS. Two different approaches were used to evaluate the fractionation: fractionation index and U/Th ratios. The resulting fractionation index values indicate low fractionation for the majority of elements evaluated, ranging between 0.8 and 1.2. The U/Th ratio suggests a higher fractionation at the beginning of the ablation process. To evaluate whether fractionation affects the quantification of glass samples by LA-ICPMS, a comparison of LA results with solution ICPMS analysis was conducted. The distribution of particle sizes during the ablation under different conditions and laser systems was also measured to evaluate the fractionation. The standard reference materials NIST 612, 610, and 1831 were analyzed in triplicate by both methods (n = 55) along with a set of 10 casework samples originating from different automobiles.