The therapeutic effect of many drugs can be improved in the form of prolonged release preparations. Prolonged action of the drug is of particular importance in the treatment of chronic diseases because it reduces the frequency of drug use which is more acceptable for both the patient and the healthcare system. Dexamethasone is a glucocorticoid used to treat a number of chronic diseases. Previous works have shown that dexamethasone in the form of an oil solution with the addition of a cosolvent has a possible useful application due to its prolonged release properties. In these works, many release parameters were examined, but there was still a need for additional verification of the method over a longer duration of the test due to the possible occurrence of interferences. Therefore, the aim of this work was to optimize the method for dexamethasone release from oil solutions on a USP II device coupled with optic fiber spectrophotometer and to examine potential interferences. Castor oil was used as a solvent due to highest dexamethasone solubility compared to other tested oils. Isopropanol and Capryol®90 as cosolvents increase solubility of dexamethasone in oil, and in addition reduce the viscosity of the solution leading to increased injectability. The assay was performed on a USPII device coupled with optical fibers that monitored the concentration of dexamethasone released from the formulations via absorption in receptor medium. The absorption profile of placebo formulations was determined and in the second part of the assay additional measurement of released dexamethasone by UPLC was introduced so that interferences during optical probes detection could be determined more precisely. The results of the study confirmed the existence of interference by optical probes detection, meaning that there is an excessive detection of released dexamethasone in the formulation with isopropanol after 36 hours and with Capryol®90 after 24 hours duration of the experiment. Interference occurs most likely due to the release of excipients from the formulations. Therefore, method of direct determination of dexamethasone release by optical fibers is reliable for analysis up to 24 hours. Furthermore, since about 80% of dexamethasone is released from both formulations after 24 hours, the method is still good enough to compare the release rate between different dexamethasone oil formulations as well as for for their initial screening.