Abstract Background Statins are used to treat and prevent cardiovascular diseases (CVDs) by reducing the total serum cholesterol concentration. Unfortunately, dose-related side effects and sub-optimal response, attributed to non-adherence amongst others, were described. Therefore, a fast and sensitive liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) method for adherence testing and therapeutic drug monitoring of all currently marketed statins and their active metabolites in human blood plasma should be developed, validated and tested for applicability. Methods Atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin, as well as ortho- and para-hydroxy-atorvastatin, lovastatin hydroxy acid and simvastatin hydroxy acid were included and several internal standards (IS) tested. Validation was performed according to the guideline of the European Medicines Agency including selectivity, carry-over, accuracy, precision, matrix effects, dilution integrity and analyte stability. Finally, applicability was tested using 14 patient samples submitted for regular toxicological analysis. Results Due to an analytical interference of atorvastatin-d5, diazepam-d5 and pentobarbital-d5 were chosen as IS for positive and negative ionization mode, respectively. All statins and metabolites fulfilled the validation acceptance criteria except for fluvastatin, which could not be quantified reliably and reproducibly, most probably due to instability. Analyses of human plasma samples revealed concentrations of statins and metabolites below the reference plasma concentrations in the case of eight patients. However, nothing was known concerning patients’ adherence and time between intake and sampling. Conclusions An LC-HRMS/MS method for identification and quantification of atorvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin and four active metabolites was successfully developed and applicability demonstrated.