PURPOSE: Presently, the majority of clinical tools to quantify deviations in patient setup during external beam radiotherapy is based on two-dimensional (2D) analysis of portal images. The purpose of this study is to develop a tool for the inspection of the patient setup in three dimensions (3D) and to validate its clinical advantage over methods based on 2D analysis in the presence of out-of-plane rotations. METHODS AND MATERIALS: We developed an interactive procedure to quantify the setup deviation of the patient in 3D. The procedure is based on fast computation of digitally reconstructed radiographs (DRRs) in two beam directions and comparison of these DRRs with corresponding portal images. The potential of the tool is demonstrated on three selected cases of prostate and parotid gland treatment where conventional 2D analysis produced inconsistent results. The measurements from 3D analysis are compared with those obtained from the 2D analysis. RESULTS: Despite application of an immobilization cast, two investigated parotid gland setups showed rotational deviations in 3D up to 3 degrees. Two-dimensional analysis of these deviations produced inconsistent results. Analysis of the selected prostate setup in 3D showed a rotational deviation of 7 degrees around the left-right axis, possibly causing displacement of the seminal vesicles toward the borders of the conformal boost fields. Using 2D analysis, this out-of-plane rotation was misinterpreted as a translation resulting in the failure to trigger the decision protocol to correct the setup after the first fraction. Using the 3D patient setup analysis procedure, an accuracy of the order of 1 mm and 1 degree (SD) could be obtained. The computation time of the interactive DRRs is of the order of 1 s on a 60 MHz PC. The complete interactive 3D analysis requires about 10 min. CONCLUSIONS: Quantification of the patient setup in 3D provides essential additional information in cases where conventional 2D analysis is inconsistent, e.g., in the presence of out-of-plane rotations or geometrical degeneracies. The speed and accuracy of the interactive 3D patient setup inspection are acceptable for use in offline clinical studies and analysis of problem cases.