Circular RNA (circRNA) is becoming an increasingly popular field of study and topic of interest. With that, there is a renewed and heightened interest in generating circular RNA for both research and clinical use. CircRNA generation began in the 1990s using in vitro chemical ligation and has since expanded out to include in vitro enzymatic synthesis along with in vivo vector-based systems. Each of these methods has distinct advantages, limitations, and different function 1ality depending on application. Throughout this chapter, we explore and summarize different methods for generating pure circRNA constructs that can be readily utilized for different applications. We highlight the steps and aspects to be considered when selecting the method to synthesize circRNA based on yield, purity, and application. Overall, this chapter aims to provide the reader with an understanding of the current methods and applications of synthesizing circRNA both in vivo and in vitro for use in research and clinical settings.