Benzodiazepines exhibit central nervous system depressive activity as well as sedative, hypnotic, and anticonvulsant properties, which enable to use them as medical treatment in anxiety, epilepsy, insomnia and alcohol withdrawal syndrome. However, from 2000s illegal benzodiazepine derivatives have started to emerge on illicit drug market as new psychoactive substances (NPSs) monitored in many countries. Analysis of both pharmaceutical drugs and NPSs from benzodiazepines group could be challenging, as usually very low concentrations need to be determined. Thus, an ultra-sensitive UHPLC-QqQ-MS/MS method was developed for simultaneous determination of 54 benzodiazepines (pharmaceutical drugs, NPS and their metabolites) and 3 z-drugs with one metabolite in biological fluid samples. The lower limit of quantification for most substances was 50 pg/mL, whereas for 17 substances as low as 10 pg/mL was achieved. Together with reduced sample volume to 100 μL it makes the developed method suitable for a sensitive multidrug toxicological analysis. Presented method was applied in routine toxicological practice as well as for the determination of benzodiazepines, z-drugs and their metabolites in 25 authentic biological fluids (blood, urine, vitreous humor and bile), both antemortem and postmortem. 19 different compounds, including benzodiazepines, their metabolites and z-drugs were determined. Antemortem blood concentrations were within 0.2-114.5 ng/mL, whereas concentrations in antemortem urine samples were 0.03-102.6 ng/mL. In postmortem specimens, concentrations ranged within 0.2-473.2 ng/mL, 0.5-94.1 ng/mL, 1.3-208.8 ng/mL and 41.5-42.0 ng/mL in blood, vitreous humor, urine and bile, respectively. The developed method is suitable for a forensic toxicology analysis, as well as clinical toxicology which is evidenced by the positive results of international proficiency tests.