Chemical reaction networks that catalyze the conversion of a reagent with high chemical potential (fuels) into lower-chemical-potential "waste" molecules are used to power the cells' molecular machinery or molecular assembly. The ATP-driven ATPase pump and GTPdriven dynamic assembly of tubulin into microtubules are prototypical examples. Inspired by such biomolecular machinery, reaction cycles that catalytically convert fuels into waste have been devised to power synthetic molecular machineries like dynamic assembly fibers driven by the hydrolysis of methylating agents or the rotation of a molecular motor catalyzed by the hydration of carbodiimides. In these reaction cycles, two chemical reactions operate simultaneously: (1) an activation reaction is the reaction between the high chemical potential (which we call fuel) and a catalyst, which activates the catalyst, and (2) a deactivation reaction that reverts the activated catalyst to its original state (Scheme 1A). The free energy released by the two reactions can be used to operate molecular machinery or to regulate selfassembly. For example, suppose the activated catalyst can self-assemble in its short lifetime. In that case, the corresponding assemblies are dynamic because their building blocks are constantly exchanged with freshly activated building blocks. These exchange dynamics result in kinetically controlled assemblies vastly different from their in-equilibrium counterparts. Similarly, if the activated catalyst can undergo a supramolecular interaction, the chemical fuel can power supramolecular machinery like pumps or motors. Our group and the Hartley group introduced a particularly powerful reaction cycle driven by the hydration of carbodiimide-based fuels (Scheme 1B). In the activation, the carbodiimide fuel activates a carboxylate-containing catalyst, for example, by converting it into its corresponding anhydride. The anhydride spontaneously hydrolyzes in the deactivation. Thus, the carboxylate catalyzes the hydration of the carbodiimide fuel to its corresponding urea waste by transiently becoming an activated catalyst in the form of an anhydride. The carbodiimide-fueled reaction cycle has gained widespread attention. It is frequently used to regulate the phase separation of complex coacervate-and oil-based droplets, the formation of macrocycles, 8, 13-14 the aggregation of nanoparticles, 15 the self-assembly of peptides into vesicles and fibers, the crystallization of amino acids, and the crosslinking of polymer networks. More recently, the cycle was recently used to drive molecular motors and regulate molecular pumps , and control DNA folding. 31 Despite its success, the carbodiimide-driven reaction cycle has drawbacks. Most prominently are side reactions that arise from the intermediate O-acylurea state-after the reaction between the carbodiimide and the catalyst, this shortlived species is formed 32 and reacts in three pathways (Scheme 1C). Firstly, it reacts with a nucleophile like a carboxylate to form the desired activated catalyst. Secondly, the O-acylurea can hydrolyze by reacting with water which means the catalyst is recovered without performing its function, i.e., inefficient fuel use. Lastly, the O-acylurea undergoes an intramolecular O-N arrangement, yielding its corresponding N-acylurea. This reaction is irreversible; thus, forming N-acylurea decreases the fuel's efficiency and irreversibly destroys the catalyst, i.e., the side reaction poisons the catalyst. The latter reaction is disastrous for chemically fueled reaction cycles. In context, even if only 5% of converted fuel yielded the N-acylurea, 99% of the catalyst would be poisoned after it undergoes 100 activation-deactivation cycles. The elegance of chemically fueled biomolecular machinery is that it can run thousands of cycles. For synthetic molecular machinery to live up to these levels, catalyst poisoning through side reactions must be understood and suppressed. Therefore, in this work, we tested how simple parameters could be varied to suppress any side reaction in the popular carbodiimide-fueled reaction cycle. The concentration and the structure of the catalyst, the structure of the carbodiimides, the presence of additives, and environmental conditions like temperature and pH were all screened using a kinetic analysis to suppress unwanted N-acylurea and increase the yield of the main product. Our work provides guidelines for using carbodiimide-fueled reaction cycles to regulate molecular function by choosing the optimal conditions. We apply these guidelines to a case study and show that the difference between optimal conditions and sub-optimal conditions can be the difference between chemically fueled assemblies or not.