The limited flexibility and time-consuming nature of the genetic manipulation procedures applicable to Trypanosoma cruzi continue to restrict the functional dissection of this parasite. We hypothesised that transformation efficiency could be enhanced if electroporation was timed to coincide with DNA replication. To test this, we generated epimastigote cultures enriched at the G1/S boundary using hydroxyurea-induced cell-cycle synchronisation, and then electroporated parasites at various time points after release from the cell-cycle block. We found a significant increase in transformation efficiency, with both episomal and integrative constructs, when cultures were electroporated 1 h after hydroxyurea removal. It was possible to generate genetically modified populations in less than 2 weeks, compared to the normal 4-6 weeks, with a 5 to 8-fold increase in the number of stably transformed clones. This straightforward optimisation step can be widely applied and should help streamline functional studies in T. cruzi.