Long-term peritoneal dialysis (PD) frequently produces morphological and functional changes of the peritoneum, which makes continuation of PD difficult. Moreover, the progression of peritoneal injury causes complications and poor prognosis. Since therapeutic treatments for peritoneal injury during PD have yet to be established, it is important to diagnose peritoneal injury as early as possible. The aim of this study was to develop a method of monitoring peritoneal function to diagnose peritoneal injury. Model rats of peritoneal injury were prepared by intraperitoneal injection of methylglyoxal (MGO) for five consecutive days. Then, marker substances of various molecular weights (phenolsulfonphthalein, fluorescein isothiocyanate-dextran (FD)-10, FD-40, FD-70, FD-2000 or tetramethylrhodamine-dextran (RD)-10) were injected into the peritoneal cavity. At 120 min after injection, the remaining amounts of all marker substances were significantly decreased in the MGO-treated rats compared with those in the vehicle-treated rats. Molecular weight dependence of the peritoneal permeability was observed. A substance with a molecular weight of approximately 10000 was found to be suitable to diagnose peritoneal injury. Moreover, coadministration of RD-10 with FD-2000 enabled us to monitor enhanced peritoneal permeability and the transfer of water simultaneously, without the recovery of whole PD fluid, even in the case of different ultrafiltration volumes. We demonstrated the usefulness of administering substances to evaluate peritoneal permeability and the transfer of water simultaneously to diagnose peritoneal injury. This study should be valuable for safe and effective PD.