Chromatographic system for simultaneous determination of levodopa, biogenic amines, and methylxanthines in food has been developed. Chromatographic column and precolumn with octadecylsilane phase and simultaneously fluorescence and DAD detectors have been used. Gradient elution with acetate buffer (pH=4.66) with acetonitrile has been applied. Examination included levodopa, norepinephrine, dopamine, normetanephrine, tyramine, and serotonin as well as caffeine, theophylline, and theobromine. Limit of detection (LOD) and limit of quantitation (LOQ) have been determined for all compounds with signal to noise ratio (S/N) equal to 3 and 10, respectively. LOD of 10 ng/mL and LOQ of 30 ng/mL for levodopa and biogenic amines as well as LOD of 70 (60) ng/mL and LOQ of 210 (180)ng/mL for methylxanthines have been determined. Authors have also developed method for simultaneous separation of all analytes from food matrix. Developed chromatographic system with sample preparation method has been applied for determination of examined compounds in cocoa products, vegetables, and fruits.