Fluorescence recovery after photobleaching (FRAP) is a well-established method commonly used tomeasure the diffusion of fluorescent solutes and biomolecules in living cells or tissues. Here afiber-optic-based FRAP (f-FRAP) system was developed, and validated using macromolecules in water andagarose gels of different concentrations. We applied f-FRAP to measure the site-specific diffusion offluorescein (NaFluo) in peritoneal membranes (PMs) on the liver, cecum, and kidney of a living rat duringperitoneal dialysis. Diffusion of fluorescein in PM varied in a time-dependent manner according to thetype of organ (DPM on Liver/DNaFluo = 0.199 ± 0.085, DPM on Cecum/DNaFluo = 0.292 ± 0.151, DPM on Kidney/DNaFluo= 0.218 ± 0.110). The proposed method allows direct quantitative measurement of the three-dimensionaldiffusion in local PM in vivo, which was previously inaccessible by peritoneal function test methods suchas peritoneal equilibration test (PET) and standardized PM assessment (SPA). f-FRAP is promising forlocal and dynamic assessments of peritoneal pathophysiology and the mass transport properties of PMs,presumed to be affected by variation of tissue structures over different organs and functional changes ofthe PM with years of peritoneal dialysis