One of the less understood branches of modern neurobiology is the (in)ability of adult mammals to regenerate their central nervous system (CNS) after injury. This is possible in species such as octopuses and lizards but is lacking in mammals as it becomes lost during development. Monodelphis domestica, the grey South American short‐tailed opossum is one of the few mammals that can fully and functionally regenerate their CNS after injury in the first two postnatal weeks, whereas most other species from the mammalian class lose their regenerative capacity soon after birth. Opossum are born very immature and continue to develop latched to the mother's belly, which therefore makes them excellent candidates for neuroregenerative and neurodegenerative research. Corticogenesis of the opossum brain is mostly postnatal, but in contrast to rodents has not been sufficiently explored. To better understand the process of corticogenesis and transition of heterogenous neural stem cells (NSC) into neurons, a specific method is required for precise quantification of their number and characterization of their expression profile. For this purpose, the isotropic fractionator (IFR) method has been modified and optimized for work with opossum tissues and under inverted fluorescent microscope, and used to identify, quantify, and classify cortical cell lines of the developing opossum brain. Data gained on fixed and homogenized cortical tissue samples has been compared with the recently established primary dissociated opossum cortical cell cultures, which also exhibit regenerative potential during different developing periods in vitro. Additionally, the robustness and precision of the IFR has been validated for opossum tissue by comparing the results with immunohistochemically labelled cortical tissue slices and primary dissociated opossum cortical cell cultures. Three timepoints of interest have been chosen for research of postnatal development of the opossum cortex. The timepoints have been previously examined on the opossum spinal cord, which gave us the cerebral timepoints in conjunction with the knowledge that the maturation process of developing spinal cords starts in cervical and ends in lumbar regions. The timepoints chosen are: postnatal day 5‐6 (P5‐6), where the regeneration is still possible; P17‐18, where the regenerative properties cease, but the brain is still not fully developed; and P30, where all the cortical structures are present, with a variety of cell lines. We successfully developed IFR for opossum tissues at various postnatal ages and for the inverted microscope, both as a one‐day and two‐day protocol variants. Additionally, we showed that many of the existing antibodies can be efficiently used on opossum due to the high protein homology between opossum and the immunogen/referent animal for which the antibodies were developed.