The objective of this paper is to develop a novel digital subtraction technique for serial intra-oral radiography, which would allow the detection of subtle variations in grey values. Digital images of the maxilla of a dried human skull and of a fresh pig mandible were acquired using intra-oral photostimulable phosphor plates (Digora FMX, Soredex, Helsinki) with an aluminium calibration stepwedge incorporated in the filmholder. Exposures were made with an X-ray tube for intra-oral radiography (Prostyle Intra, Planmeca, Helsinki). During pilot testing, parameter settings were adapted to reach an optimal contrast. Exposures were repeated within a 1-week interval to determine the test-retest reliability of the development. After in vitro and in vivo testing, the exposure technique and software development were used to evaluate its applicability in a pilot clinical case. Although parameter settings remained stable during the in vitro studies, the clinical exposures yielded non-linear digital images, thus, not readily suitable for data acquisition and comparison of the regions of interest. To allow further analysis, image processing was carried out using self-developed software for semi-automated linearisation and optimised contrast normalisation. This processing significantly increased the precise quantisation of jawbone density and the assessment of subtle bone density changes in arbitrarily selected regions of interest of in vivo exposures. The clinical applicability of the technique is demonstrated in a pilot case. It was demonstrated that minute densitometric deviations could be detected. The present technique and image processing may allow the quantification of jawbone density.