The ubiquitin-proteasome pathway plays a crucial role in the regulation of many physiological processes and in the development of a number of major human diseases, such as cancer, Alzheimer, Parkinson, diabetes etc. As a new target, the study on the proteasome inhibitors has received much attention recently. Three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using comparative molecule field analysis (CoMFA) and comparative molecule similarity indices analysis (CoMSIA) techniques were applied to analyze the binding affinity of a set of tripeptide aldehyde inhibitors of 20S proteasome. The optimal CoMFA and CoMSIA models obtained for the training set were all statistically significant with cross-validated coefficients (q2) of 0.615, 0.591 and conventional coefficients (r2) of 0.901, 0.894, respectively. These models were validated by a test set of 8 molecules that were not included in the training set. The predicted correlation coefficients (r2) of CoMFA and CoMSIA are 0.944 and 0.861, respectively. The CoMFA and CoMSIA field contour maps agree well with the structural characteristics of the binding pocket of β5 subunit of 20S proteasome, which suggests that the 3D-QSAR models built in this paper can be used to guide the development of novel inhibitors of 20S proteasome