To establish a method for determining tubeimoside I content in Bolbostemma paniculatum (Maxim.) Franquet; and to investigate the anti-proliferative mechanisms of tubeimoside I on K562 cells. HPLC was employed. Column: RP-C18 (4.6 × 250 mm 5 μm); mobile phase: methanol-water (V:V=68:32); DAD detection wavelength: 214 nm; temperature: room temperature. Effect of tubeimoside I on K562 cell proliferation was determined by MTT assay; and apoptosis rate was measured by flow cytometry. Tubeimoside I exhibited linearity within a 0.408-2.040 mg/mL range (r=0.9996). Average recovery was 98.98% (n=5), with RSD=1.82%. MTT assay showed that treatment with different doses of tubeimoside I for 12 and 24 h could all reduce the viability of K562 cells. Flow cytometry with PI staining revealed that tubeimoside I could induce K562 cell apoptosis within the test concentration range. Compared to the control group, apoptosis was positively correlated with drug concentrations, showing marked dosedependence. The present method is rapid, simple and accurate, which can be used for determining tubeimoside I contents in Bolbostemma paniculatum (Maxim.) Franquet and its preparations. Tubeimoside I has an anti-proliferative effect on K562 cells.