The simultaneous determination of the monoamines dopamine (DA), noradrenaline (NA) and serotonin (5-HT) in in vivo microdialysis samples remains challenging because of the low extracellular neurotransmitter levels in different brain regions, specific sample characteristics, and the quest for high temporal resolution and a multi-target strategy in neuropharmacological research. A fast and sensitive microbore (1.0 mm i.d. column) UHPLC method coupled to electrochemical detection (ECD) is developed by means of design of experiments with the emphasis on sufficient retention of NA within an acceptable total analysis time. Indeed, NA is the earliest eluting compound and often interferes with the broad solvent front originating from the sample matrix. The sensitive UHPLC-ECD assay (LLOQ of 100 pM for NA and 150 pM for DA and 5-HT) with an analysis time of 8 min for standard solutions and 20 min for in vivo microdialysis samples originating from rat hippocampus, prefrontal cortex and striatum, is validated applying accuracy profiles. The combination of in vivo microdialysis and microbore UHPLC-ECD has shown to be particularly suitable for future contributions to neuropharmacological research on the monoaminergic system.