Preclinical Single-Photon Emission Computed Tomography (SPECT) is an imaging tool that has gained importance over the years thanks to technological improvements and to the development of novel radiotracers to image a wide variety of biologically relevant targets. Preclinical SPECT scanners offer improved spatial resolution and sensitivity compared with their clinical counterparts and allow in vivo experimentation in animal models of disease. Research using several radiotracers targeting neurological and oncological applications has provided important insight into several diseases and advanced drug development. With the advent of radiotheranostics, the role of preclinical SPECT has become even more significant for providing radiation dosimetry estimates.