Adoption of soybean that is resistant to 2,4-D will result in more use of glyphosate plus 2,4-D premixes and tank mixtures. Preliminary whole-plant greenhouse assays confirm most Palmer amaranth populations found in Indiana are glyphosate-resistant (GR), and some biotypes exhibit tolerance to 2,4-D amine. Dose–response experiments were conducted to determine the level of glyphosate resistance and 2,4-D amine tolerance in four Palmer amaranth biotypes. A premix formulation of glyphosate plus 2,4-D choline was also evaluated. The R1, R2, and R3 biotypes were 31- to 66-fold more resistant to glyphosate (R:S ratio) than the S1 biotype based on the herbicide dose to cause 90% mortality (LD90). The maximum POST rate of the premix formulation of Enlist Duo® labeled in ‘Enlist®’ soybean is 2,195 g ae ha−1. When separated by active ingredient, the maximum POST rate of Enlist Duo® is equivalent to 1,141 and 1,054 g ae ha−1 of glyphosate and 2,4-D choline, respectively. In the absence of glyphosate, the maximum rate of 2,4-D (1,054 g ae ha−1) in the premix formulation of Enlist Duo® controlled S1, R2, and R3 biotypes, but failed to control all plants from the R1 biotype. Estimates for LD90 showed the R1 biotype was 3-fold more tolerant than the S1 biotype to 2,4-D amine. However, no plants survived the 1,155 g ae ha−1 (600 g ae ha−1 of glyphosate plus 555 g ae ha−1 2,4-D) treatment with the premix formulation of glyphosate plus 2,4-D choline. Overall, results from this experiment suggest GR Palmer amaranth that also exhibits increased tolerance to 2,4-D amine will be difficult to control with glyphosate or 2,4-D alone, but can be controlled POST with Enlist Duo® at lower than labeled field rates (1,618 to 2,195 g ae ha−1).