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Dental shade determination and seamless integration of restorative work in the oral cavity are challenging and important tasks in the everyday clinical dental practice. The aim of this in-vitro study was to evaluate comparatively the capability of software-based color analysis of mobile phone photography, with the spectrophotometric and visual methods for dental shade determination. Visual shade determination of the incisal, middle, and cervical thirds parts of ten extracted human teeth was performed using the Vita Classical and Vita 3D Master stock shade guides. Shade determination of the thirds of each tooth was performed using the Vita Easyshade spectrophotometer. Subsequently, photographs of each tooth were captured using a mobile phone camera. Color charts were produced using an in-house image processing technique, and the tooth color captured by the mobile phone photography was matched to the shade guides. The results show that the camera-based method had better agreement with the spectrophotometric and visual methods when the Vita Classical shade guide was employed. Software-based color analysis of mobile phone photography should be further explored for its use as an affordable potential tool for increasing objectivity and accuracy in dental shade determination.