Mirabegron is the first registered β3-adrenoceptor agonist for treatment of overactive bladder as an alternative to antimuscarinics. Previously four liquid chromatography-tandem mass spectrometry assays were published to determine mirabegron and eight metabolites (M5, M8, M11-M16) in human plasma. In order to support paediatric development, the assays were further optimized to reduce the required blood volume and increase the sensitivity. The assays were miniaturized by using 96-well supported liquid extraction plates (mirabegron, M5, M16) or 96-well mixed-mode cation exchange solid phase extraction plates (M8, M11-M15) and a more sensitive MS-system was used. For the analytes, up to fivefold increase in assay sensitivity was achieved. The required blood sample volume was reduced from 10 to 2 mL for each timepoint. Validation demonstrated that the assays were accurate, precise and selective in the determination of mirabegron and metabolites. The assays were successfully applied to evaluate the pharmacokinetics of mirabegron in a paediatric population.