Background: Prosopis glandulosa Torr. (Fabaceae) is native to Africa, America, and Asia. In India, it is abundantly found in southern parts. The plant is reported to contain polyphenols, flavonoids, mesquitol-catechin dimers, indolizidine alkaloids, and triterpenes, imparting antitumor, antimicrobial, anti-infective, and anti-parasitic activities. Aim: The present study deals with the isolation and characterization of flavonoid C-glycosides from P. glandulosa Torr. leaves. Materials and Methods: P. glandulosa leaves were subjected to hot aqueous extraction at 90°C—100°C to yield crude aqueous extract (PGA). PGA was fractionated and then subjected to chromatography over Diaion® HP-20 column and semi-preparative high-performance liquid chromatography to provide flavonoid C-glycosides. Results: We have identified and characterized vicenin-2/isomer and schaftoside (s) from P. glandulosa leaves through liquid chromatography-electron spray ionization-mass spectrometry/mass spectrometry (LC-MS/MS) and nuclear magnetic resonance. LC-MS/MS chromatogram displayed peaks at retention time of 9.69 and 10.37 with m/z 594 and major fragments at m/z 473 [M-H-120]− (loss of glucose unit) for “vicenin-2/isomer.” Interestingly, five major peaks were observed with retention times of 10.75, 11.17, 11.92 (major), 12.92, and 13.32 corresponding to the same mass of m/z 564 with major fragments at m/z 473 [M-H-90]− (loss of arabinose unit) and 443 [M-H-120]− (loss of glucosyl unit), together termed as “schaftosides.” Conclusion: To the best of our knowledge, this is the first report on flavonoid C-glycosides (vicenin-2/isomer and Schaftosides) from P. glandulosa leaves which may act as chemomarker(s) for the standardization and quality control of this plant.