The metabolic profiling of biological fluids is important in understanding the various biochemical processes in the human body. The content of aromatic metabolites, including microbial ones, in the blood and cerebrospinal fluid, can provide essential information reflecting infectious processes, both systemic and in the central nervous system. A sensitive method with protein precipitation and sample concentration using ultra-high-pressure liquid chromatography-tandem mass spectrometry was proposed and subsequently validated to determine the number of aromatic metabolites of phenylalanine, tyrosine, and tryptophan in the blood serum and cerebrospinal fluid at 2.0-3.7 × 103 nmol/L. Reference values of 4-hydroxybenzoic, 3-(4-hydroxyphenyl)propionic, and indole-3-carboxylic acids were measured in the blood serum of healthy donors (n = 48). Profile of eleven phenyl- and indole-containing acids was revealed in the serum samples (n = 29) of the patients with long-term sequelae of severe brain damage and in the cerebrospinal fluid samples (n = 29) of the post-neurosurgical patients using different sample preparation methods to measure analytes in a wide (nmol/L and μmol/L) concentration range. Statistically significant differences in concentrations of most analytes were detected in serum samples of patients compared to healthy donors (p ≤ 0.03) and in concentrations of 3-phenyllactic, 3-(4-hydroxyphenyl)lactic, indole-3-lactic, and indole-3-carboxylic acids in cerebrospinal fluid samples of patients with signs of secondary bacterial meningitis compared to those without signs of secondary bacterial meningitis (p ≤ 0.027).