Differences in botulinum neurotoxin manufacturing, formulation, and potency evaluation can impact dose and biological activity, which ultimately affect duration of action. The potency of different labeled vials of incobotulinumtoxinA (Xeomin; 50 U, 100 U, or 200 U vials; incobotA) versus onabotulinumtoxinA (BOTOX; 100 U vial; onabotA) were compared on a unit-to-unit basis to assess biological activity using in vitro (light-chain activity high-performance liquid chromatography (LCA-HPLC) and cell-based potency assay (CBPA)) and in vivo (rat compound muscle action potential (cMAP) and mouse digit abduction score (DAS)) assays. Using LCA-HPLC, incobotA units displayed approximately 54% of the protease activity of label-stated equivalent onabotA units. Lower potency, reflected by higher EC50, ID50, and ED50 values (pooled mean SEM), was displayed by incobotA compared to onabotA in the CBPA (EC50: incobotA 7.6 0.7 U-mL; onabotA 5.9 0.5 U-mL), cMAP (ID50: incobotA 0.078 0.005 U-rat; onabotA 0.053 0.004 U-rat), and DAS (ED50: incobotA 14.2 0.5 U-kg; onabotA 8.7 0.3 U-kg) assays. Lastly, in the DAS assay, onabotA had a longer duration of action compared to incobotA when dosed at label-stated equivalent units. In summary, onabotA consistently displayed greater biological activity than incobotA in two in vitro and two in vivo assays. Differences in the assay results do not support dose interchangeability between the two products.