The herbicide 2,4-D is available in distinct formulations in terms of selectivity to non-target species, and in terms of drift and volatilization potentials. Thus, the objective of this work was to evaluate the selectivity and effect of 2,4-D formulations (amine salt and choline) applied at different developmental stages of barley. The experiment was installed in the field in randomized blocks, in a 2 × 4 + 1 factorial scheme, with four replications. Factor A comprised the formulations of 2,4-D (amine salt and choline), and factor B the crop phenological stage (before tillering start, at full tillering, first node and booting), plus a weeded control treatment. The 2,4-D amine showed higher phytotoxicity when applied before tillering and at the first node. On the other hand, 2,4-D choline, when used prior to tillering start, showed the highest phytotoxicities. The yield components showed positive results when using the amine salt before and at tillering, while in the first node stage, and in booting, the choline salt stood out. The use of 2,4-D amine in tillering and choline in the first node provided the highest grain yield of barley.