Dextromethorphan (DEX) is an antitussive agent used in many cough and cold medications, and dextrorphan (DOR) is its metabolite. Owing to their similar structures, optimization of the condition for the chromatography approach, which is in common use for determination, is both demanding and time-consuming. This paper describes a methodology that combines excitation-emission matrix fluorescence spectra with second-order calibration, and was applied to simultaneously and directly determine DEX and DOR contents in plasma samples.