In vivo microdialysis has been used in preclinical pain research for more than a decade. This valuable tool allows correlations between nociceptive behavior and neurotransmitter release in pain-related CNS sites. However, several methodological issues must be considered to adequately interpret microdialysis data. Thus, the aim of this review is to describe key considerations, potential pitfalls, and important control experiments. We focus on animal experiments which evaluate the effects of noxious stimulation on CNS neurotransmitter release, particularly those that address clinically relevant problems in patients with long-lasting painful conditions.