Assessing the efficacy of botulinum neurotoxin (BoNT) _in vivo_ is essential given the growing number of BoNT products used in the clinic. Here, we evaluated the dynamic weight bearing (DWB) test for sensitivity to paralytic effects of BoNT-A following intramuscular administration. The toxin was administered into the gastrocnemius lateralis as a single bolus or into the gastrocnemius lateralis and medialis as two boluses. The effects of BoNT-A in DWB were compared to those in the compound muscle action potential (CMAP) and the Digit Abduction Score (DAS) tests. Female Sprague-Dawley rats received an acute, intramuscular (i.m.) injection of BoNT-A1 (0.1, 1, 10 pg/rat) into the right gastrocnemius muscle, while the left received vehicle. The DWB and CMAP tests were performed one-two days after the injection in order to detect the onset of sub-maximal BoNT-A activity. Both tests were preceded by the DAS test. BoNT-A produced dose-related reductions in both the weight-bearing and surface-bearing outcomes of up to 60% while showing moderate activity in the DAS. BoNT-A effects in the DWB test were well-aligned with those in the CMAP test, which showed dose-dependent reductions in CMAP amplitude and the area under the curve (AUC; up to 100%) as well as increases in latency (up to 130%). The efficacy of BoNT-A in DWB and CMAP was more pronounced with two boluses. Thus, the DWB test can be used to assess the properties of BoNTs following i.m. administration. It can be used to assess the candidate therapies and is more ethical than the mouse lethality assay.