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Abstract Agrobacterium‐mediated transient expression methods are widely used to study gene function in both model and non‐model plants. Using a dual‐luciferase assay, we quantified the effect ofAgrobacterium‐infiltration parameters on the transient transformation efficiency ofCatharanthus roseusseedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre‐ and post‐infiltration dark incubation and is less sensitive to theAgrobacteriumgrowth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven‐ to eight‐fold while a dark incubation pre‐ and post‐infiltration increased transformation efficiency by five‐ to 13‐fold.Agrobacteriumin exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in ourAgrobacterium‐infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6‐fold in raw firefly luciferase (FLUC) and rawRenillaluciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation ofAgrobacteriuminfiltration inC. roseusseedlings will facilitate the study of this important medicinal plant and will expand the application ofAgrobacterium‐mediated transformation methods in other plant species.