Designing a feasible in vitro enzyme cascade is a challenging task that is often preceded by optimization steps. Computational methods can increase the efficiency of the optimization process by providing a deeper insight into the system. In this study, bottlenecks of a farnesyl pyrophosphate‐producing in vitro enzyme cascade were determined by a combination of in silico modeling and wet‐lab experiments. ATP and the precursor‐isomerizing enzyme isopentenyl diphosphate δ‐isomerase were identified as limitations of the in vitro enzyme cascade.