The protocol for image-guided microbeam radiotherapy (MRT) developed for the Australian Synchrotron's Imaging and Medical Beamline (IMBL) is described. The protocol has been designed for the small-animal MRT station of IMBL to enable future preclinical trials on rodents. The image guidance procedure allows for low-dose monochromatic imaging at 50 keV and subsequent semi-automated sample alignment in 3D with sub-100 µm accuracy. Following the alignment, a beamline operation mode change is performed and the relevant beamline components are automatically aligned for the treatment (pink) beam to be delivered on the sample. Here, the small-animal MRT station, the parameters and procedures for the image guidance protocol, as well as the experimental imaging results using phantoms are described. Furthermore, the experimental validation of the protocol using 3D PRESAGE® dosimeters is reported. It is demonstrated that the sample alignment is maintained after the mode change and the treatment can be delivered within the same spatial accuracy of 100 µm. The results indicate that the proposed approach is viable for preclinical trials of small-animal MRT.