In order to investigate the diffusion behavior of poly(ethylene imine) (PEI) into keratin fibers, cross‐sectional samples of bleached white human hair treated with PEI were prepared. We were successful in developing a method for analyzing the diffusion behavior of PEI into human hair, which to our knowledge is a first. The diffusion pattern of PEI into human hair, which cannot be determined by optical microscopy, can be determined by our method. After the treatment, the cross‐sectioned hair samples were dyed with Orange II and the cross‐sectional intensity scans were measured at a wavelength of 487 nm (λmax of Orange II) with a microspectrophotometer. In our method, the diffusion pattern of PEI at pH 11.1 showed Fickian type characteristics. This suggests that the diffusion coefficient of PEI is essentially independent of the PEI concentration. By calculating the diffusion coefficient from the PEI concentration profile, the diffusion coefficient of PEI [number‐average molecular weight (Mn) = 300 and 600] into the bleached human hair was found to be on the order of 10−10 cm2/s. In addition, the diffusion coefficient of PEI (Mn = 600) with urea added increased twofold in comparison with that of PEI without urea added. This experiment demonstrated that urea acts as a penetration accelerator for PEI. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 65–71, 2005