Multi-slice computerized tomography (MSCT) is considered to provide superior image quality. We defined a data acquisition protocol for high-resolution (HR) temporal bone imaging using MSCT and assessed its impact on data acquisition and post-processing (PP). The data acquisition protocol was defined in cadaveric phantom studies performed by MSCT and subsequently applied to 38 patients referred for temporal bone assessment. The parameters image quality and diagnostic value of MSCT data were assessed for the cross-sectional source images as well as for 2-dimensional (2D) reformations and 3-dimensional (3D) reconstructions by 3 radiologists by comparison with incremental HR scans of 17 patients with suspected middle ear disorders. The data acquisition protocol yielded HR images with an excellent detail resolution and a comparable image quality of cross-sectional scans and related orthogonal reformations. MSCT achieved higher scores for image quality and diagnostic value (p < 0.001, t-test) than incremental HR CT with regard to both 2D and 3D reconstructions. MSCT improves the image quality of HR cross-sectional scans as well as that of 2D and 3D PP techniques in petrous bone imaging. The radiation exposure of the eye lenses is increased by MSCT as gantry angulation is not yet possible in the helical scan mode.