Tobacco has proven to be a promising alternative for the production of recombinant therapeutic proteins and offers numerous advantages over other plants as a host system. However, the recovery and purification steps needed to obtain a protein at high recovery and purity have not been well investigated. In this study, a process was developed to purify a model acidic protein, recombinant beta-glucuronidase (rGUS) from transgenic tobacco leaf tissue, in three main steps after extraction: polyelectrolyte precipitation, hydrophobic interaction chromatography (HIC), and hydroxyapatite chromatography (HAC). Using this three-step process, up to 40% of the initial rGUS activity could be recovered to near homogeneity as judged by SDS-PAGE. This work demonstrates that acidic recombinant proteins expressed in tobacco may be purified to high yield with high purity in a minimal amount of steps that are suitable for scale-up. Furthermore, the general steps used in this process may suggest that a wide variety of acidic recombinant proteins may be purified in a similar manner from transgenic tobacco or other leafy crops.