There is growing evidence for nitric oxide (NO.) being involved in cell signaling and pathology. Much effort has been made to elucidate and characterize the different biochemical reaction pathways of NO.in vivo. However, a major obstacle in assessing the significance of nitrosated species and oxidized metabolites often remains: a reliable analytical technique for the detection of NO. in complex biological matrices. This chapter presents refined methodologies, such as chemiluminescence detection and flow injection analysis, compared with adequate sample processing procedures to reliably quantify and assess the circulating and resident NO(.) pool, consisting of nitrite, nitrate, nitroso, and nitrosylated species.