The purpose of this study was to establish a modified method of three-compartment model analysis that minimized the sampling frequency. A Caco-2 permeability assay was performed on ten structurally diverse compounds with passive diffusion. A three-compartment model was analyzed by a conventional method and a method with fewer sampling points, called the simplified method, using concentration–time profiles in the donor, intracellular, and receiver compartments. The concentration–time profiles in all compartments were well described by the conventional method. The calculated unbound fraction of intracellular ( fu ₂) and apparent permeability coefficient ( P _app) were 0.0107–1.22 and 0.886-146 × 10⁻⁶ cm/s, respectively. The simplified method also described the concentration profiles in the compartments of all ten compounds except one, ibuprofen. The difference in values calculated by the simplified method compared to the conventional method was between −7 and 7% for fu ₂ and between −6 and 42% for P _app. These results suggested that the parameter values from the simplified method were comparable with those from the conventional method. The simplified method may be a promising approach to improve the throughput of three-compartment model analyses of Caco-2 permeability assays in the early stages of drug discovery.