A chromatographic system is developed for the separation and determination of levodopa, biogenic amines, and their metabolites from the catecholamines group: dopamine (DA), epinephrine (E), normetanephrine (NMN), metanephrine (MN), 3,4- dihydroxyphenylacetic acid (DOMA), 3-metoxy-4-hydroxyphenylglycol (MHPG), and homovanillic acid (HVA); and indoloamines group: serotonin (5HT) and 5-hydroxyindole-3-acetic acid (5HIAA) in urine. The limit of detection (LOD) and limit of quantitation (LOQ) are determined for all compounds with signal-to-noise ratio (S/N) of 3 and 10, respectively. LOD 10 (ng/mL) and LOQ 30 (ng/mL) are determined for L-DOPA, DOMA, E, NMN, DA, MN, and MHPG, as well as LOD 8 (ng/mL) and LOQ 24 (ng/mL) for HVA, 5HT, and 5HIAA. A fluorescence detector is used. Gradient elution with acetate buffer (pH = 4.66) with methanol is applied. In urine samples from patients treated with levodopa, the following concentrations (µg/mL) of analytes are determined: L-DOPA 3.73–46.80, DOMA 1.43–28.43, E 0.83–13.57, NMN 2.58–8.81, DA 24.07–62.11, MN 0.89–66.20, MHPG 6.72–63.64, 5HT 22.96–95.27, 5HIAA 1.45–14.77, and HVA 0.21–15.07.