Microbial carboxylases and decarboxylases play important roles in the global carbon cycle and have many potential applications in biocatalysis and synthetic biology. The widespread family of reversible UbiD-like (de)carboxylases are of particular interest because these enzymes are active against a diverse range of substrates. Several characterized UbiD enzymes have been shown to catalyze reversible (de)carboxylation of aromatic and aliphatic substrates using the recently discovered prenylated FMN (prFMN) cofactor, which is produced by the associated family of UbiX FMN prenyltransferases. However, discovery and investigation of novel UbiD (de)carboxylases are delayed by our limited knowledge and the experimental complexities associated with FMN prenylation and UbiD activation resulting in the production of inactive recombinant UbiD enzymes. Therefore, there is a need for developing robust methods for efficient in vitro and in vivo FMN prenylation and UbiD activation for heterologous production of active UbiD enzymes. In this chapter, we present two protocols for in vitro and in vivo FMN prenylation and UbiD activation under aerobic conditions using enzyme cascades with regenerating systems and recombinant E. coli cells.