When used for intra-operative imaging of residual basal cell carcinomas (BCCs), reflectance confocal microscopy (RCM) is limited to detection of relatively large tumors. Small tumors remain hidden in the surrounding bright dermis. Fluorescence confocal microscopy (FCM) may improve the sensitivity for detecting small tumors. Fluorescein enhances cell cytoplasm contrast in fluorescence confocal images, but has had limited clinical impact on imaging BCCs in vivo because there is a lack of a well-defined protocol (concentration and application time) that can be effectively used for intraoperative imaging. We conducted an ex vivo study, using discarded tissue from Mohs surgery and a benchtop FCM with 488nm wavelength for excitation and 521nm detection for imaging Concentrations of 6, 0.6 and 0.6 mM with immersion times of 5, 15, 30, and 60 seconds were repeatedly tested (total of 76 specimens).. The 0.6 mM and immersion time of 60 seconds showed that cellular cytoplasm can be labeled with controlled saturation and without leaving the yellow color on the surface of the tissue. Initial results show that, fluorescein may enhance cellular structures contrast relative to other normal dermal structures, improving the detection of small BCCs. This study provide an optimized set of parameters for subsequently testing of topical application in vivo for intraopertive imaging of BCCs.