Over the past decade, time-of-flight (TOF) and Orbitrap™ (Thermo Fisher Scientific) mass spectrometers have aroused a growing interest, and we observed a progressive replacement of low-resolution mass spectrometers (LRMS) by high-resolution mass spectrometers (HRMS). Thanks to high mass accuracy, they provide an unambiguous identification and allow structural elucidation of unknown compounds. Furthermore, they offer new powerful acquisition modes based on untargeted screenings dependent or independent of previous information. These advances are of particular interest in the field of toxicology, and we observed an increasing number of HRMS presentations at scientific meetings, corresponding original papers, and review articles dealing with application HRMS. The aim of this study is to compare HRMS with respect to LRMS in toxicology, to detail and explain the physical and technological principles on which the TOF and the Orbitrap mass analyzers are based, the data acquisition modes and data processing methods. Finally, three applications in toxicology are detailed to highlight the interest of this technology in toxicology.