Purpose:Verification of the mechanical isocenter position is required as part of comprehensive quality assurance programs for stereotactic radiosurgery/radiotherapy (SRS/SRT) treatments. Several techniques have been proposed for this purpose but each of them has certain drawbacks. In this paper, a new efficient and more comprehensive method using cine-EPID images has been introduced for automatic verification of the isocenter with sufficient accuracy for stereotactic applications.Methods: Using a circular collimator fixed to the gantry head to define the field, EPID images of a Winston–Lutz phantom were acquired in cine-imaging mode during 360° gantry rotations. A robust matlab code was developed to analyze the data by finding the center of the field and the center of the ball bearing shadow in each image with sub-pixel accuracy. The distance between these two centers was determined for every image. The method was evaluated by comparison to results of a mechanical pointer and also by detection of a manual shift applied to the phantom position. The repeatability and reproducibility of the method were tested and it was also applied to detect couch and collimator wobble during rotation.Results:The accuracy of the algorithm was 0.03 ± 0.02 mm. The repeatability was less than 3 μm and the reproducibility was less than 86 μm. The time elapsed for the analysis of more than 100 cine images of Varian aS1000 and aS500 EPIDs were ∼65 and 20 s, respectively. Processing of images taken in integrated mode took 0.1 s. The output of the analysis software is printable and shows the isocenter shifts as a function of angle in both in-plane and cross-plane directions. It gives warning messages where the shifts exceed the criteria for SRS/SRT and provides useful data for the necessary adjustments in the system including bearing system and/or room lasers.Conclusions: The comprehensive method introduced in this study uses cine-images, is highly accurate, fast, and independent of the observer. It tests all gantry angles and is suitable for pretreatment QA of the isocenter for stereotactic treatments.