We investigated the changes (increase or decrease in peak intensity) in the expression of plasma proteins in spontaneously diabetic WBN/Kob rats that were with complicated diabetic nephropathy, to determine multiple biomarkers in the plasma of diabetic rats. The present study using surface enhanced laser desorption/ionization time of flight mass spectrometry (SELDI-TOF-MS) demonstrated that six peaks at mass/charge ratios (m/z) of 4678, 4732, 4808, 9058, 9323, and 9465, among approximately 80 peaks per spectrum in the 2000—10000 Da mass range, had increased peak intensities with the development or progression of diabetic nephropathy in plasma of spontaneously diabetic WBN/Kob rats as compared with those of normal Wistar rats. Administration of the Kampo medicine Hachimi-jio-gan was effective at reducing the expression of diabetic nephropathy but not at reducing blood glucose levels. It also improved the increased levels of these plasma proteins. Other biomarker peaks at m/z 5067, 5279, 7598, and 7917 were not affected by Hachimi-jio-gan administration. Further study will be needed to identify these positive biomarkers and to evaluate the relationship between the efficacy and expression patterns of the plasma proteins in greater detail. The expression patterns of proteins and molecular-related ions revealed that several proteins in plasma may be involved in the development and/or progression of diabetic nephropathy in WBN/Kob rats and the efficacy of Hachimi-jio-gan. This study using ProteinChip technology may provide a useful basis in the search for multiple biomarkers in plasma for the diagnosis of disease and therapeutic evaluation of Kampo medicines.