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Gleditsia caspica Desf. (Caesalpinaceae) is traditionally used to treat diverse diseases. This research paper aims to investigate anti-proliferative properties and phytochemical compounds of methanolic extract from the pods of G. caspica. MTT test was used to measure in vitro anti-proliferative effect of the soxhlet-extracted methanolic extract of G.caspica pods with the concentration ranging from 25-1000 μg/ml against A549 cell line. The extract was applied to SPE, HPLC preparative system, and spectral analysis (1HNMR, 13CNMR, and UV) for fractionation, purification, and identification of phytoconstituents, respectively. The results indicated that the extract prevented proliferation of A549 cell line dependent on time and concentration. After 48 h and 72 h, there was a significant decrease in cell viability initiated from 100 and 500 (μg/ml) concentrations in comparison to untreated control. Phytochemical assessment of the extract resulted in two alkaloids, locustoside A and saikachinoside A, and four flavonoids quercetin-3,7-diglucoside, luteolin-4',7–diglucoside, quercetin-3-glucoside, and quercetin. This experiment was an activity-guided phytochemical study. In prior studies, quercetin and quercetin-3-β-D-glucoside showed in vitro inhibitory effect on proliferation of A549 cell line. However, the cytotoxic properties of the other identified compounds have not been reported. Cytotoxic properties of G.caspica was previously attributed to saponins, while isolated flavonoids and alkaloids could be responsible for the extract’s cytotoxic effects. This paper first explored cytotoxic activity of methanolic extract from the pods of G.caspica on A549 cell line and identified flavonoids and alkaloids of this plant. [ABSTRACT FROM AUTHOR]