Wastewater-based epidemiology (WBE) is an innovative tool that, unlike traditional epidemiological approaches, is capable of providing real-time profiling of community health and lifestyle along with emerging trends and changes in pattern usage of drugs in wastewater. By using human urinary excreted indicators, so-called “biomarkers”, WBE provides estimates at population level. Therefore, the choice and the evaluation of suitable biomarkers of exposure to drugs is of fundamental importance for the public health monitoring. Moreover, since many drugs are chiral, the investigation on enantiomeric profiling of chiral biomarkers provides a new dimension to WBE. To aid enantiomeric profiling in WBE, sensitive enantioselective methodologies are required.In this thesis, two novel multiresidue analytical methods based on chiral liquid chromatography coupled to mass spectrometry were developed and validated.The first method investigated the main human biomarkers for the detection of illicit drugs of abuse and potentially abused licit drugs. New biomarkers were investigated, such as mephedrone, PMA and all MDMA’s metabolites. Furthermore, a case study on mephedrone posed the basis for a novel approach towards biomarker selection in estimation of human exposure to chiral drugs with limited metabolism data. As a result, mephedrone was a suitable biomarker due to its stability in wastewater. In addition, some of its metabolites were also proposed as potential candidates for mephedrone use.The second method explored biomarkers of quinolones’ use, as they represent one class of antibiotics with rising concern in antibiotic resistance. The most comprehensive panel of quinolones’ biomarkers was considered for the first time in WBE studies.Both methodologies were applied to wastewaters from eight locations in Europe allowing the first pan-European studies on enantiomeric profiling of chiral biomarkers. Key findings of this research included: the detection of high mephedrone loads only in the UK, thus indicating human consumption; the detection of HMMA, a MDMA metabolite, as a suitable indicator of MDMA consumption and the determination of different synthetic production routes of methamphetamine across Europe. With regards to quinolones’ biomarkers, higher ofloxacin loads were found in Southern European cities along with differences in enantiomeric fraction with respect to Northern ones. Moreover, ofloxacin’s metabolites showed ofloxacin use and ulifloxacin was found as a result of prulifloxacin consumption. Therefore, enantiomeric profiling led to an understanding of: (i) new patterns of emerging drugs of abuse, (ii) changes in patterns of classical illicit drugs and (iii) quinolones with the verification of the origin of drug residue, potency of abused drug and its synthetic route and (iv) quinolones’ metabolic profiles. Moreover, the simultaneous determination of quinolones’ biomarkers in European samples allowed for the verification of spatial and temporal trends of quinolones’ use and the occurrence of their resistance genes. This proof-of-concept research will facilitate further advances in the WBE field.