Abstract There is a truly vast quantity of research articles and textbooks, aimed at a variety of audiences, on cytochromes P450. However, a large amount of specialized terminology has become associated with these enzymes, which can be daunting to those new to the field. The aim of this chapter is to give a brief overview of the functions and importance of cytochromes P450 with particular emphasis on their roles as tailoring enzymes in natural product biosynthetic pathways. Differences between the biosynthetic enzymes and their catabolic counterparts are highlighted. Assays used to investigate substrate binding to cytochromes P450 are described using TxtE, a recently discovered unique nitrating enzyme involved in thaxtomin A biosynthesis, as an example.