Cotinine (COT), trans-3'-hydroxycotinine (OH-COT), cotinine-N-β-glucuronide (COT-GLUC), and trans-3'-hydroxycotinine-O-β-glucuronide (OH-COT-GLUC) are excreted in urine following the intake of nicotine (NIC), and, as such, they have been detected in sewage. Thus, they also constitute convenient biomarkers for NIC tracing through the sewage epidemiology approach at the local scale. Such estimation requires granting a good stability of the target biomarkers in sewage. However, it was found that glucuronides are not stable, particularly in the case of OH-COT-GLUC, which could render variable concentrations of COT, OH-COT, and their glucuronides, depending on sampling and storage time or temperature. Thus, an enzymatic deconjugation with β-glucuronidase was optimized. With the optimized method, after enzymatic deglucuronization, the limits of quantification obtained were in the range of 0.2-1 μg L(-1), relative standard deviations were <10%, and the trueness in terms of recovery was in the 95%-112% range. The application of the method to composite sewage samples collected during 1 week in three different years in Santiago de Compostela (Galicia, Spain) showed COT and OH-COT concentrations of 0.3-1.9 μg L(-1) and 1.0-3.3 μg L(-1), respectively. Thereby, the average NIC consumption derived was in the 1.7-1.9 mg per day and person range, being comparable to those derived from tobacco sales statistics.