Abstract: In this study, a high-throughput screening method was developed based on ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF MS) for the simultaneous screening of 180 prohibited substances in children’s cosmetics. The chromatographic separation conditions and mass spectrometry scanning mode were optimized. A screening database for the 180 prohibited substances was constructed using Library View database software, according to the retention time, exact mass of the precursor ion, and MS/MS spectrum of each compound. Chromatographic separation was performed on a Kinetex^® F5 column (100 mm×3.0 mm, 2.6 μm) with gradient elution, using 2 mmol/L ammonium formate aqueous solution (containing 0.1% formic acid) and 2 mmol/L ammonium formate methanol-acetonitrile (1:1, V/V, containing 0.1% formic acid) as the mobile phases. The eluent from the column was detected using information-dependent scanning (IDA) in both the positive and negative ion modes of electrospray ion sources. The Library View software was used to identify target compounds by matching the acquired sample data against the screening database. The sample pretreatment procedures were optimized separately for cream and liquid oil samples. The optimal extraction solvents were 50% acetonitrile for cream and 50% methanol for liquid oil, with an optimal extraction time of 40 min. Method validation was performed for the established approach. The relative standard deviations (RSDs) for the two sample matrices ranged from 1.8% to 10%, with limits of detection (LODs) ranging from 0.01 to 0.5 μg/g. The matrix effects for most tested compounds were less than 20%. The application of this high-resolution mass spectrometry-based screening database has significantly improved the efficiency of daily work and the capability for safety risk monitoring of illegal additives in children’s cosmetics. It provides technical support for the scientific regulation of cosmetics and is of great importance for ensuring the quality and safety of children’s cosmetics. Currently, this screening method is only applicable to cream and liquid oil samples. Future work will focus on optimizing pretreatment methods for other sample matrices and continuously expanding the number and variety of compounds in the database. The enhanced screening database can be applied not only to the screening of illegally added substances in children’s cosmetics, but also extended to other cosmetic categories, thereby providing a strong safeguard for the safe use of cosmetics.