Estimations, made by the World Health Organization (WHO), state that 10% of the medical products in low- and middle-income countries are substandard or falsified (SF). Among them, antibiotics and antimalarials are the most commonly reported since 2013. Besides the fact that falsification is a crime, the worldwide use of poor quality antimicrobials could result in treatment failures, stronger antimicrobial resistance and even the promotion of the emergence of superbugs. Therefore, simple and accurate analytical methods are necessary, which are capable to detect and quantify a wide range of antimicrobials in suspected illegal products. In this work, a screening and a quantification method using ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS) and diode array detection (UHPLC-DAD), respectively were developed and validated. These methods could be used for routine analysis and enable a more in-depth characterization of SF-antimicrobials. According to their popularity as SF-antimicrobials, 31 antibiotics, 3 antibacterial agents, 1 antifungal agent and 1 beta-lactamase inhibitor, covering eleven different antibacterial classes, were selected. The UHPLC-MS screening method with gradient elution is able to selectively detect these 36 compounds within 18 min (including wash and equilibration step). It was validated for sensitivity, selectivity and matrix effects. Within an analysis time of 32 min, the UHPLC-DAD method could quantify 32 compounds (4 showed insufficient UV absorbance) and resulted in sufficient selectivity, necessary since some SF-antimicrobials may include more than one antimicrobial component. This quantification method was validated for the positive hits found during screening tests of suspected illegal samples. This resulted in a validation set of 11 antimicrobials and 1 beta-lactamase inhibitor. The ''total error'' approach in accordance with the validation requirements of ISO-17025 was employed for the validation. 57 real-life illegal samples, seized by inspectors from the Belgium Federal Agency for Medicinal and Health Products (FAMHP), were analyzed using the two described methods. About half of them were not compliant and some samples that contained clavulanic acid showed a serious reduction in the amount of this molecule (in one sample only 14% of the claimed dosage was found). These quality issues might be attributed to either poor manufacturing, storage or transportation conditions.