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Inborn errors of dopamine and serotonin metabolism are diseases caused by deficiencies in enzymes belonging to metabolic pathways. The specific diagnosis of these inborn illnesses is based on the identification and quantification of biomarkers in cerebrospinal fluid (CSF), especially: 5-hydroxy-tryptophane (5-HTP), 5-hydroxy-indol-acetic acid (5-HIAA), 3-ortho-methyl-DOPA (3-OMD), homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylglycol (MHPG). In the present work, we propose a novel ultrahigh performance liquid chromatography (UHPLC) method coupled to fluorescence detection (FD) to quantify simultaneously the five dopamine and serotonin metabolites. This method efficiently separates the five molecules in less than 10 min. A complete validation of the proposed method was performed in terms of accuracy, linearity, precision, and lower limit of quantification (LLOQ). Depending on the compound, the obtained LLOQs are between 1 nM and 5 nM, thus allowing to measure concentrations as low as in CSF samples. We also verified the method applicability by analyzing 10 CSF samples in triplicates. The obtained results showed satisfactory repeatability and an ability of this method to clearly distinguish healthy samples from pathologic samples, hence, demonstrating, the method suitability for diagnosing inborn errors of dopamine and serotonin metabolism. Therefore, the proposed UHPLC-FD method appears as a reliable alternative to the current gold standard for the quantification of these biomarkers, which is based on UHPLC coupled to electrochemical detection (ECD).