Wastewater can provide a wealth of epidemiologic data on common drugs consumed and on health and nutritional problems based on the biomarkers excreted into community sewage systems. One of the biggest uncertainties of these studies is the estimation of the number of inhabitants served by the treatment plants. Twelve human urine biomarkers 5-hydroxyindoleacetic acid (5-HIAA), acesulfame, atenolol, caffeine, carbamazepine, codeine, cotinine, creatinine, hydrochlorothiazide (HCTZ), naproxen, salicylic acid (SA) and hydroxycotinine (OH COT) were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to estimate population size. The results reveal that populations calculated from cotinine, 5-HIAA and caffeine are commonly in agreement with those calculated by the hydrochemical parameters. Creatinine is too unstable to be applicable. HCTZ, naproxen, codeine, OH COT and carbamazepine, under or overestimate the population compared to the hydrochemical population estimates but showed constant results through the weekdays. The consumption of cannabis, cocaine, heroin and bufotenine in Valencia was estimated for a week using different population calculations. (C) 2016 Elsevier B.V. All rights reserved.