Gap junctional intercellular communication has been shown to be involved in the carcinogenesis process. Gap-FRAP (Fluorescence Recovery After Photobleaching) technique could be used to estimate gap junctions functionality and their potential involvement for distinguish normal and cancer cells. We developed a sensitive microspectrofluorimetric method to characterize the kinetics of fluorescence recovery after photobleaching. We used a positive control cell line (fibroblast), a negative control (FaDu) and an intermediate (KB) head and neck cancer cell lines. Our results indicate that using our approach, the fluorescence kinetic profiles remain comparable with the method widely used by confocal microscopy.