The procedure of the determination of cetrimide and chlorohexidine diiodide in polymer microparticles was proposed and verified. The described procedure combines the spectrophotometric method to determine chlorohexidine diiodide and the non-aqueous titration to determine the sum of nitrogen bases. To verify the ability of the proposed procedure to yield reliable results, the determinations of repeatability and accuracy were performed. The results confirmed the suitability of the proposed procedure for the indicated purpose. Using the procedure, samples were successfully analysed during the development of the new antiseptic powder containing antiseptic drugs in a biodegradable polymer.