Analysis of biomarkers in wastewater is increasingly seen as an important tool for valuation of health, nutrition and use of various substances by humans. Some biomarkers are used for population estimates, since the actual values of population size in cities can differ significantly from official counts, which inevitably leads to errors in assessing the impact of various factors to people calculated per capita. 5-hydroxyindole-3-acetic acid (5-HIAA) is the main metabolite of serotonin, which is excreted by urine and can be used as a biomarker for population estimates. The determination of this metabolite is more preferred than the detection of serotonin itself in terms of correctness of the results. A technique of extraction and subsequent quantitative determination of 5-HIAA in wastewater by high-performance liquid chromatography in combination with tandem mass spectrometric detection was developed. Poroshell Hilic column was used as a stationary phase which has an alternative selectivity compared to traditional C18 columns. Developed procedure is characterized by low detection limits (0.2 μg/liter) and good selectivity. The conditions for liquid-liquid extraction of 5-HIAA from wastewater were selected. This technique provides reliable estimation of the concentration of 5-HIAA in wastewater.