Nearly 2 decades have passed since initial studies demonstrated a relationship between plasma (or serum) levels of the tricyclic antidepressant drugs and clinical response. Since then, therapeutic monitoring of antidepressant drugs has become routine in many laboratories and the use of these measures is becoming a routine part of psychiatric practice. New uses for antidepressant measurements have evolved and guidelines for proper use of these measures need to be updated to take advantage of more recent findings. Continuing studies in psychiatry have also prompted technical advances in methodology that has undergone predictable evolution to robust techniques for analysis of most antidepressants. However, the diversity of methods for therapeutic drug monitoring, issues of cross-reactivity and assay interferences, and confusion over sample collection techniques still contribute to difficulties in obtaining and using serum concentration data. Differences between techniques specifically designed for therapeutic monitoring of antidepressant drugs and those designed for toxicology or emergency drug screening are not clear and lead to misunderstandings between laboratory personnel and psychiatrists. This article updates and discusses current guidelines that form the basis for using serum level measurements of antidepressant drugs and reviews salient pharmacologic, clinical, and analytical issues that must be taken into account if therapeutic drug monitoring data are to be used for optimal clinical benefit.