Carotenoid cleavage products, apocarotenoids, are biologically active compounds exerting important functions as chromophore, hormones, signaling molecules, volatiles, and pigments. Apocarotenoids are generally synthesized by the carotenoid cleavage dioxygenases (CCDs) that comprise a ubiquitous family of enzymes. The activity of plant CCDs was unraveled more than 20 years ago, with the characterization of the maize VP14, the first identified CCD. The protocol developed to determine the activity of this enzyme in vitro is still being used, with minor modifications. In addition, in vivo procedures have been developed during these years, mainly based on the exploitation of Escherichia coli cells engineered to produce specific carotenoid substrates. Further, technological developments have led to significant improvements, contributing to a more efficient detection of the reaction products. This chapter provides an updated set of detailed protocols suitable for the in vitro and in vivo characterization of the activities of CCDs, starting from well-established methods.