In the study, three aristololactams were isolated from Piper wallichii and determined simultaneously by a HPLC method using a C18 column with a gradient elution of methanol and water. The content ranges of the target compounds in twelve batches of Piper wallichii were 0.100 - 0.110 mg/g dry weight (cepharanone B), 0.198 - 0.208 mg/g dry weight (aristolactam AII), and 0.047 - 0.059 mg/g dry weight (aristololactam AIIIa), respectively. Their anti-proliferative effects in HK-2 cells were determined by MTT assay and cells membrane damage were assessed by LDH release assay. Results indicated that the test compounds showed a time- and dose-dependent cytotoxicity in HK-2 cells, and the values of IC50 were cepharanone B > aristolactam AII > aristololactam AIIIa.