The diffusion coefficient (D) of peptide and protein drugs needs to be determined to examine the permeability through biological barriers and to optimize delivery systems. In this study, the D values of fluorescein isothiocyanate (FITC)-labelled dextrans (FDs) and peptides were determined and the permeability through a porous membrane was discussed. The observed D values of FDs and peptides, except in the case of insulin, were similar to those calculated based on a relationship previously reported between the molecular weight and D of lower-molecular-weight compounds, although the molecular weight range was completely different. The observed D value of insulin was between the calculated values for the insulin monomer and hexamer. The permeability of poly-lysine and insulin through the membrane was determined and the observed values were compared with predicted values by using the relationship between molecular weight and D and an equation based on the Renkin function. The observed permeability of insulin through the membrane was between that of the predicted permeability for the insulin monomer and hexamer. For the permeation of insulin, the determination of D was useful for estimating the permeability because of the irregular relationship between molecular weight and D. The methodology used in this study will be useful for a more quantitative evaluation of the absorption of peptide and protein drugs applied to mucous membranes.