Diffusion coefficients of proteins in chromatographic media are important parameters for the rational design of stationary phases and purification schemes. In contrast to free diffusion, intraparticle diffusion is hindered by the porous structure of the media. Direct intraparticle diffusion analysis (IDA) is a novel approach for the determination of intraparticle protein diffusion coefficients. IDA is based on the evaluation of spatially and temporally resolved intraparticle concentration profiles. To prevent adsorption and to study diffusion only, the chromatographic media are investigated in underivatized form. With IDA, intraparticle concentration profiles are measured in a microcolumn by confocal laser scanning microscopy (CLSM). From this dynamic data, the diffusion coefficients are determined by parameter estimation, using a spheric diffusion model. The boundary condition is given by the measured protein concentration in the bulk phase. IDA is applied to determine intraparticle diffusion coefficients of seven different proteins in Sepharose 6 FF. The results show excellent congruence of experimental data and simulation results. Moreover, the determined diffusion coefficients lie well within the range of data published in the literature. Given that the material in question allows optical analysis, IDA is a general approach for studying protein diffusion in porous particles and is easily adapted to different proteins, solution conditions and stationary phases.