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Tricyclic antidepressants have been prescribed for the treatment of depression and other disorders since their discovery in the 1950s but have been replaced in recent decades by newer drugs with more favorable side effect profiles. However, for some patients and conditions, tricyclic antidepressants remain the drug of choice. A fast, sensitive, and robust UPLC-MS/MS method for the monitoring of amitriptyline, nortriptyline, imipramine, doxepin, and desipramine in human urine has been developed using a pre-defined and systematic method development approach. The method was developed using sub-2p.m particle technology, providing a state-of-the-art alternative to older methods. Total cycle time was 2.5 min. Human urine samples (200 mu L) were prepared using an Oasis (R) WCX mu Elution solid-phase extraction plate, which provided good recovery for all analytes (>92%) and low matrix effects (absolute matrix effects <10%). Standard curves were linear over the range 0.02-250 ng/mL with r(2) values >0.994. The method was evaluated against current FDA guidelines and was applied to the analysis of patient samples, including an assessment of incurred sample reanalysis (ISR). (C) 2013 Elsevier B.V. All rights reserved.