A sensitive and specific LC-MS/MS method was developed for simultaneous determination of aloe-emodin, rhein, emodin, chrysophanol and physcion and their conjugates in rat plasma. The lower limit of quantitation of each anthraquinone was 0.020-0.040μm. Intra-day and inter-day accuracies were 90.1-114.3% and the precisions were <14.6%. The matrix effects were 104.0-113.2%. The method was successfully applied to a pharmacokinetic study in rats receiving a rhubarb extract orally. The area under the concentration-time curve (AUC0-t) and peak concentration (Cmax) of free aloe-emodin and emodin in rat plasma were much lower than those of rhein. The amounts of chrysophanol and physcion were too low to be continuously detected. After treating the plasma samples with β-glucuronidases, each anthraquinone was detectable throughout the experimental period (36h) and showed much higher plasma concentrations and AUC0-t. The free/total ratios of aloe-emodin, rhein and emodin were 6.5, 49.0 and 1.7% for Cmax and 3.7, 32.5 and 1.1% for AUC0-t, respectively. The dose-normalized AUC0-t and Cmax of the total of each anthraquinone were in the same descending order: rhein>emodin>chrysophanol>physcion>aloe-emodin. These findings reveal phase II conjugates as the dominant in vivo existing forms of rhubarb antharquinones and warrant a further study to evaluate their contribution to the herbal activity. © 2013 John Wiley & Sons, Ltd.