Wastewater-based epidemiology (WBE) relies on the principle that traces of compounds, which a population is exposed to or consume, are excreted unchanged or as metabolites in urine and/or feces, and ultimately end up in the sewer network. Measuring target metabolic residues i.e. biomarkers in raw urban wastewater allows identifying the exposure or use of substances of interest in a community. Up to date, the most popular application of WBE is the estimation of illicit drug use and studies have been made mainly across Europe, which has allowed estimating and comparing drug use in many European cities. However, until now a comprehensive study applying WBE on the most frequently consumed illicit drugs has not been performed in South American countries. In this work, we applied this approach to samples from Colombia, selecting two of the most populated cities: BogotĂĄ and Medellin. Several biomarkers were selected to estimate drug use of cocaine, cannabis, amphetamine, methamphetamine, MDMA (ecstasy), heroin and ketamine. Composite samples (24-h) were collected at the corresponding municipal wastewater treatment plants. Sample treatment was performed at location by applying solid-phase extraction (SPE). Before SPE, the samples were spiked with appropriate isotope labeled internal standards. In parallel, samples (spiked with the analytes under study at two concentration levels) were also processed for quality control. Analysis of influent wastewater was made by liquid chromatography-tandem mass spectrometry, with triple quadrupole analyzer. Data shown in this paper reveal a high use of cocaine by the population of the selected Colombian cities, particularly from Medellin, while the use of other illicit drugs were low. The relevance of using quality control samples, particularly in collaborative studies, as those presented in this work, where research groups from different countries participate and where the samples had to be shipped overseas, is highlighted in this paper.