Display omitted •Human insulin and six therapeutic analogues are resolved on a mixed mode column.•Development of two separation methods: for binary and quaternary pump delivery system.•SPE cleanup of formulations with insulin recoveries from 80 to 100% is optional.•Identification and quantitation of insulins is possible using simple HPLC-UV method.•Methods were validated and can be used for screening of pharmaceutical raw materials. Human insulin and six most used therapeutic analogues are very similar in terms of retention on a reversed-phase column. Thus, the LC methods prescribed in the European Pharmacopoeia monographs for insulin and insulin analogues include many similar separation methods, which tend to be time consuming when separating individual products of insulins or are inadequate when handling a mixture. In this study, we present a simple, robust, versatile and accessible HPLC-UV separation method for identification and quantification of human insulin and its analogues in one run. The simultaneous separation and detection is possible by fine-tuning the mobile phase properties that affect the separation mechanism on a mixed mode column combining anion exchange and reversed-phase characteristics. Also developed was a simple and effective SPE sample cleaning procedure with insulin recoveries ranging from 80 to 100% for all analogues. On the other hand, the concentration of major excipients such as phenol and m-cresol fall below 1%. The two developed and validated separation methods differ in their compatibility with the use of a quaternary or binary pump, thus enabling sample characterisation independent of the HPLC solvent delivery system. The methods are compatible with the use of a mass spectrometric detector for an indisputable identification.