There has been an increasing focus on development of new routes of dexamethasone administration to provide targeting of the drug for local action, and also to reduce systemic side effects and avoid the first pass metabolism through the liver. For topical administration of dexamethasone is necessary to develop an appropriate therapeutic system that would enable sustained and controlled release of the drug. The aim of this study was to develop and compare the analytical methods for in vitro release testing of the active substance dexamethasone from nanoparticle-based therapeutic system using three apparatuses for in vitro release testing of topical formulations (imersion cell, vertical diffusion cell and dialysis bag membrane method), taking into account the physiological conditions at the point of application of these therapeutic systems. Diffusion of the drug occured across polyethersulfone membrane, or across dialysis cellulose membrane, which demonstrated low drug adsorption and low diffusional resistance. After an initial lag phase the amount of drug released became proportional to the square root of time. The slope in the linear portion of the release curve was used as a measure of release rate. The imersion cell method was found to have the best discriminatory power and repetability for in vitro release testing of dexamethasone from nanosuspension. Although the dialysis bag membrane method revealed sufficiently sensitive to the tested parameters, great variability (RSD) in the results was observed. The vertical diffusion cell did not show sufficient sensitivity to this type of samples due to the small surface of the membrane across which the diffusion of the therapeutic agent occurs.