Transient plant tissue transformation in Nicotiana tabacum is a valuable technique in molecular biology applications. N. tabacum is highly susceptible to transformation and sustains significant levels of heterologous gene expression. The infiltration process includes an initial phase of T-DNA expression without integration into the genome that can be used for different purposes such as gene expression analysis, quantification of protein accumulation, and metabolite production. To overcome the limitations of conventional infiltration methods, a vacuum infiltration protocol for young N. tabacum plants was developed, allowing simultaneous infiltration of multiple plants. Here, we show the application of the transient agroinfiltration method using the 35S:RUBY plasmid, encoding a biosynthetic pathway for the red-purple betalain pigment to facilitate the visualization of the transformed tissue over time.