The penetration of various amino acids and dipeptides through porcine skin was measured in vitro in this work to examine the effect of lipophilicity and molecular weight of the compounds on the permeability of passive diffusion. Experimental results indicated that the effect of lipophilicity is a more dominant factor than the molecular weight. An equation was also developed to accurately predict the permeability of amino acids and dipeptides based on the lipophilicity of penetrants as follows: log K(p) = -2.73 + 0.38ln K -0.036(ln K)2 with R2 of 0.85 for the regression, where K, the lipophilicity, is represented by the partition coefficients of amino acids and dipeptides between the PEG/Na2SO4 aqueous two-phase system. In particular, the successful prediction of partition coefficients of dipeptides by Chen's method was used to further predict the dipeptides' permeability for transdermal delivery. The experimental results were deemed satisfactory.