Pyrrolizidine alkaloids (PAs) and tropane alkaloids (TAs) are natural toxins which can be found in honey and are recognized for their hepatoxic and neurotoxic properties. Given honey's widespread consumption as part of the human diet, ensuring its safety is mandatory [1]. Therefore, an accredited method [2] was employed to detect 33 PAs and 21 TAs in honey, based on ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS) with a quadrupole linear ion trap (Q-TRAP) system. Honey samples were extracted using a solvent mixture of methanol and water acidified with formic acid. In a comprehensive analysis of 33 Czech and Spanish honey samples from diverse botanical origins, it was found that among the 21 Czech samples, 6 tested positive for PAs and TAs. However, only two of them exhibited quantifiable residues of echimidine (1.4 µg/kg), lycopsamine (197 µg/kg), and intermedine (140 µg/kg). Among the 12 analyzed Spanish honeys, 5 tested positive for nine PAs (intermedine, lycopsamine, heliotrine, europine, seneciphylline, senecionine, senecivernine, echimidine and lasiocarpine). Total PA concentrations in Spanish honeys ranged from 0.4 to 65.1 µg/kg. Contrastingly, none of the monitored TAs were detected in these analyzed samples. The new Commission Regulation 2023/915 does not currently include maximum residue limits for these compounds in honey. Instead, it outlines limits solely for pollen-based food supplements. In light of the demonstrable potential health hazards associated with these toxins, it is expected that accurate analytical methods and more extensive investigations for PAs and TAs in honey-based products will be developed to enhance their safety for potential consumers.