Single-photon emission computed tomography (SPECT) can be used to measure the three-dimensional (3D) distribution of inhaled aerosol deposition in the lungs. This is of value in evaluating and optimizing drug delivery by inhalation. 3D imaging has the advantage over planar scintigraphy of giving better data on the distribution of deposition within the lung. There are a variety of different methods of acquisition and analysis of the data, which makes interpretation of results difficult to compare between centers. This article describes a standardized protocol that aims to overcome this problem. Although not being completely prescriptive, it presents the key recommendations that are necessary to ensure consistency. In addition to radiolabel validation, these are (i) having some form of accountability of the activity measurements as quality control, (ii) producing quantitative images by performing attenuation and preferably scatter correction, (iii) defining volumes of interest and calculating parameters in the manner described, and (iv) describing in any report the details of technique used. When carefully used, SPECT imaging is able to produce high-quality quantitative data of the 3D distribution of drug deposition within the lungs. By establishing a standardized protocol, results of 3D imaging of the deposition of orally inhaled aerosols using SPECT should be more comparable, which should enhance collaborations between centers and insure that this form of imaging becomes acceptable to the regulatory authorities.