Generalized lymphatic anomaly (GLA) is a congenital, non-neoplastic vascular anomaly characterized by diffuse or multifocal involvement and infiltration of multiple organs. Most GLA cases harbor “hotspot” somatic missense single nucleotide mutations in the PIK3CA gene, whereas a minority involve mutations in other genes. Recently, rapamycin and its homologs have shown encouraging results in the treatment of alterations affecting the PI3K/AKT/mTOR pathway. Therefore, the present work aimed to expand the immunophenotypic characterization of the lymphatic lesion at the tissue level and to evaluate the in vitro viability, migration, and angiogenic capacity of endothelial cells freshly isolated from a PIK3CA-mutated GLA sample, following treatment with alpelisib, rapamycin, dactolisib, and buparlisib. We detected the p.Gln546Lys amino acid substitution in the PI3Kca helical domain in GLA-derived endothelial cells. Then, by immunohistochemical analysis, we documented altered expression of mTOR and its well-characterized downstream substrates, which regulate growth, proliferation, migration, angiogenesis, and survival of ECs. Finally, pharmacological treatment reduced GLA-EC functional properties and down-modulated the hyperactivated PI3K downstream substrates. Overall, our results suggest that routine immunohistochemistry, integrated with phospho-protein analysis and next-generation sequencing, might improve histopathological classification of vascular anomalies and facilitate the development of personalized targeted therapies, particularly in recurrent or surgically challenging cases.