Betablockers are one of the most frequently used medications in cardiology. They can lead to fatal drops in blood pressure and heart rhythm disturbances. Death is functional, and poisoning with this group of drugs can be difficult to detect. The liquid–liquid extraction (LLE) method developed using ethyl acetate at pH 9 successfully identified 18 β-blockers in human blood. The method’s limit of quantification (LOQ) was in the range of 0.1 to 0.5 ng/mL. No carryover of substances between samples was detected, and no interfering ion current signals were observed in the biological samples at the retention times of the compounds or internal standards. All compounds had a coefficient of determination (R2) above 0.995. Intraday and interday precision (RSD%) and accuracy (RE%) for low and high QC levels were within 1.7–12.3% and −14.4 to 14.1%, respectively. Very good recovery (80.0–119.6%) and matrix effect (±20.0%) values were achieved for all compounds. In addition, fragmentation spectra were collected for all the examined substances, and high-resolution spectra were presented for landiolol and metipranolol, because they are not available in commercial HRMS spectra databases. The developed method was applied in authentic postmortem samples.