A screening and confirmatory method for the determination of 13 antibiotic groups including 91 substances was developed for muscle samples and validated using ultra-performance liquid chromatography (UPLC) coupled to time-of-flight mass spectrometry. Optimisation of the parameters was performed for every analyte and personal compound database library was created, comprising the mass spectra for following collision energies; 0, 10, 20 and 40 eV. Validation plan scheming and calculation of the parameters was performed using validation software, which included following performance characteristics; selectivity, trueness, precision, decision limit (CCα), detection capability (CCβ) and relative matrix effect. High resolution mass spectrometry operated in a narrow mass window and resolving power of 50,000 FWHM (2 GHz). Accurate mass measurements with mass accuracy below 10 ppm for all analytes were obtained in spiked muscle samples. The validation experiments showed good selectivity and specificity. The method was validated in the measurement range from 0.5 to 1000 μg/kg, where a specific analyte mixed standard solution was prepared depending on the defined maximum residue limit (MRL) or lowest calibration level (LCL). For screening analysis, detection capabilities (CCβ) below the set MRL values or at LCL concentration levels were defined. CCβ, as the concentration above which the sample can be considered suspect, ranged from 0.63 to 207.8 μg/kg. For confirmation of the analyte, CCα was calculated at LCL or at the MRL, where it ranged from 0.38 to 14.3 μg/kg for unauthorised substances and from 13.8 to 1492.6 μg/kg for authorised substances. Relative within-laboratory reproducibility standard deviation ranged from 5.1 to 34.2%. Satisfactory z-score values in the range from −0.87 to 1.88 were obtained as a result of participating in four proficiency tests organized by European Union Reference Laboratories. Display omitted •Screening and confirmatory HRMS method for 91 antibiotics.•Wide validation range around the established residue limits.•CCβ and CCα as parameters for screening and confirmatory analysis.•Satisfactory proficiency results as confirmation of good method performance.