A simple and sensitive method for the simultaneous determination of plasma concentrations of five polyamines in normal and Hepatoma‐22 mice, and mice treated with Mylabris and Acanthopanax senticosus was developed by HPLC‐ESI‐MS. Male Kunming mice were divided into nine groups, a control group (inoculation without treatment), a positive group (Cyclophosphamide), treatment groups [Mylabris (4, 8, 16 mg/kg), Acanthopanax senticosus (6, 12, 24 g/kg)] and a normal group (without inoculation). Twenty‐four hours after the last administration, plasma samples were collected. The derived polyamines were separated on a C18 column by a gradient elution using methanol–water with excellent linearity within the range from 2.5 to 1000 ng/mL. Polyamines were confirmed as useful biochemical markers of hepatoma. The differences in anti‐cancer therapeutic efficacy between Mylabris and Acanthopanax senticosus might contribute to the variability of polyamine levels in vivo. This HPLC‐ESI‐MS method was successfully applied to investigate the relationship between polyamines and cancer in mice and might be a useful method to test the activity of potential anti‐tumor drugs. Copyright © 2012 John Wiley & Sons, Ltd.