Polycyclopropanated (POP) compounds show promise as fuels as their energy density can be greater than jet and rocket fuels in current use, but realizing their full potential requires significant development. This protocol guides the production of polycyclopropanated fatty acids in Streptomyces; POP production in another host remains to be demonstrated. This method can serve as a baseline for further development of POP as well as other polyketide products. For complete details on the use and execution of this protocol, please refer to Cruz-Morales et al. (2022).¹.