Haplophyllum pedicellatum Bunge (Rutaceae) is a perennial herbaceous plant indigenous to Central Asia from which alkaloids, flavonoids, and coumarins have been previously isolated. This study investigated the aerial part of the plant collected during flowering and fruiting near Aktash, Samarkand District, Republic of Uzbekistan. Air-dried raw material (6.31 kg) was extracted with methanol, and the extract was separated into fractions using water, CHCl3, and ethyl acetate. Column chromatography over silica gel with gradient elution (hexane, ethylacetate, methanol) was used to isolate compounds. Alkaloids (skimmianine, γ-fagarine, acetylevoxine, haplopine, evoxine, glycoperine, glucohaplopine), lignans (suchilactone, justicidin B, diphyllin), and a flavonoid glycoside (limocitrin 7-O-β-D-glucopyranoside) were identified by methods including TLC, mixed mp, UV, IR, 1D/2D PMR, 13C NMR, HSQC, HMBC, ROESY, and acid hydrolysis. A total of 11 compounds were obtained, among which 7 components (including the alkaloids acetylevoxine, glycoperine, glucohaplopine, all three isolated lignans, and the flavonoid glycoside) were first observed in the aerial part of H. pedicellatum. The cytotoxicity of glycoperine and limocitrin 7-O-β-D-glucopyranoside on HeLa and HCT-116 cancer cells was evaluated using WST-1 reagent. Limocitrin 7-O-β-D-glucopyranoside exhibited moderate cytotoxic activity with IC50 values of 50 μM (HeLa) and 58.5 μM (HCT-116), while glycoperine showed very low cytotoxicity for both cell lines with IC50 > 100 μM.