Fluorescence recovery after photobleaching (FRAP) is an established technique for determining solute diffusivity in biological tissues. Recently, we proposed a novel fluorescence photobleaching method, based on two independent analyses of the video-FRAP image series: the fast Fourier transform (FFT) and the Karhunen-Loève transform (KLT). In this study, the principles of this method are illustrated, and its optimization is discussed. Computer simulated FRAP experiments were used for the optimization of the choice of frequencies used in FFT analysis. The sensitivity of the technique to image noise contamination was evaluated. Additionally, the method was experimentally validated by characterizing fluorescein diffusivity in human annulus fibrosus. The results were compared to diffusivity data obtained by other methods. This study provides new insights into the optimization of data analysis of video-FRAP experiments. Furthermore, the experimental results verified the applicability of the method for the determination of solute diffusivity in biological media.