Despite the introduction of newer antidepressants, tricyclic antidepressants are still widely used for the treatment of major depression, chronic pain and anxiety. And is the most leading cause of drug-induced death in many patients. Monitoring their blood/plasma concentration is therefore a priority in forensic toxicology. In this study, a quick and rapid method for simultaneous detection and quantification of three TCAs (desipramine, nortriptyline and amitriptyline) in whole blood was developed and validated on HPLC-UV under a gradient elution, using a C18 column and a mobile phase mixture of ACN (35% v/v) and a pH 3.5, 50mM phosphate buffer (65% v/v). The evaluated validation parameters were satisfactorily accepted in accordance with ICH and FDA guidelines. The determination coefficients were more than 0.99 within the tested concentration range (0.1–1mgL -1 ), LOD and LOQ were between 0.0027-0.0051 and 0.27-0.051 respectively, Accuracy expressed as % Er was found to be between 0.1-7% and precision expressed as %CoV was found to be between 2-10%. All compounds were satisfactorily determined in one single injection within 20 min. sample pre-treatment was optimised on three sets of cartridges (C2,C18 and Strata-X) on a solid phase extractions, with C18 giving the best recovery for all analytes ranging from 60-105%. The developed method proved to be suitable for routine work and it was used to successfully analyse forensic blood samples.