Background: Ovarian tissue cryopreservation and transplantation are becoming increasingly important issues for preserving female fertility as shown by recent successes in restoring ovarian activity and even fertility. Primordial follicle content before transplantation is a key issue for success. We investigated two novel methods to detect primordial follicles in human ovarian cortical tissue strips. Materials and Methods: The first method used the fluorescent mitochondrial stain rhodamine 123 in combination with laser scanning confocal microscopy (LSCM). The second used a simple stereomicroscopic method with glass-bottomed dishes for detecting primordial follicles in cortical ovarian tissue strips. Potential toxic effects of R123 and of the exposure to confocal laser were investigated in a mouse ovarian allograft model. Results: Follicles were visible as white spots in thin cortical strips using LSCM in single and fast scanning at low magnification, allowing a fair estimation of the number of primordial follicles present. Using the second method, ovarian follicles were also visible using glassbottom dishes under the stereomicroscope, although tissue thickness and density were limiting factors of its success. Discussion: Follicles can be visualized in human cortical ovarian strips with R123 in combination with LSCM. Stereomicroscopy using glass-bottomed dishes and transmitted illumination is a simple alternative method and has the advantage of allowing further safe clinical use of the analysed tissue.