Aim: To develop an HPLC-UV method for the determination of atomoxetine (ATM) in human serum with simple sample preparation and higher automation.Methods: After protein precipitation (PP) of the serum sample, 750 μL supernatant was injected into the HPLC system with two gradient pumps and a six port switching valve to do on-line solid-phase extraction by a reverse-phase C18 column (Waters XBridge, 4.6 mm×30 mm, 5 μm) and a gradient mobile phase consisted of acetonitrile and phosphate buffer.