Perinatal illicit drug consumption has become a critical concern because of associated adverse mental and physical outcomes in the child. Drug screening in meconium can identify prenatal drug exposure beginning at the 12th week of gestation and therefore is gaining interest in pediatric care. In our laboratory, meconium is routinely analysed with an EN15189 accredited UPLC-MS/MS Multi-Target-Screening method (MTS) on a Waters Acquity UPLC connected to a Xevo TQ-XS. The method targets a panel of 65 licit and illicit drugs at LLOQs between 0.1 and 2 ng/g meconium. In addition, a commercial library based “known-unknown” screening is performed with the Bruker ToxTyper™ (TT) system. In this study, we describe our analytical findings from routine to compare the performance of these two methods. A 200 μL aliquot of a homogenized meconium suspension (1 mL H2O per 100 mg meconium) is fortified with deuterated internal standards for MTS ( n = 65) or TT (n = 12) and subsequently hydrolysed with β-glucuronidase B-ONE (Kura Biotech) for 30 min at room temperature. Thereafter, a salting out assisted-liquid/liquid-extraction is conducted by adding 800 μL 10 M ammonium acetate/acetonitrile (1/3, v/v). After centrifugation 300 μL from the organic supernatant is transferred into glass vials and evaporated into 15 μL ethylene glycol under N2 at 45 °C. The residue is then diluted in mobile phase A and measured by MTS and TT method applying a library search in the Bruker (980 entries) and Maurer/Wissenbach/Weber library (2nd edition 2019, > 4000 entries). The majority of the samples (58%) came from the German states of Bavaria, Saxony and Thuringia. From 479 consecutive routine samples analysed, 270 (56%) contained illicit substances and 145 (30%) contained prescription/OTC drugs. The remaining 64 (14%) samples presented negative in both methods. With MTS, 41 out of 65 possible analytes were found, among which amphetamine ( n = 124, 26%) and methamphetamine ( n = 101, 21%) revealed a high positive rate. With TT however, only 79 (16%) and 84 (18%) samples were positive, resulting in an agreement of 64% and 83%, respectively. THC and its metabolites OH-THC and THC-COOH showed different detection rates with MTS and TT; 119 and 2 THC positive samples (agreement 1.7%), 152 and 85 11-OH-THC positive samples (agreement 56%) and 144 and 43 THC-COOH positive samples (agreement 30%). The positive rates of MTS for other abuse relevant drugs were: morphine 5.8%, codeine 4.4%, ketamine 4.8% and cocaine 1.5%. Agreement with TT was: 57% for morphine, 91% for codeine, 61% for ketamine and 43% for cocaine. TT never found a substance from the MTS panel alone. Based on the comprehensive databases, additional 52 drugs could be reported by TT. In 16 cases, pethidine and piritramide were detected; all other substances were prescription drugs in some cases related to pregnancy. The concentrations of most parent drugs observed in meconium were generally lower than their main metabolites, such as methadone, buprenorphine, cannabis, cocaine, ketamine. Our work revealed that TT had a lower detection rate than MTS with an agreement of < 80% for most of the detected parameters. Furthermore, the majority of the additional drugs identified by the TT approach were of no clinical relevance. Cannabis, methamphetamine and amphetamine had the highest prevalence, which can be rated as a southeast Germany phenomenon. Interestingly, the cannabis metabolite OH-THC had the highest positive rate (32%) and the highest concentration of the 3 cannabinoids. The positive rates for drugs of abuse underline the usefulness of meconium for drug testing. The best future strategy would be to extend the MTS method rather than continue with the cost-intensive TT method.