A positive response to scalene muscle block (SMB) is an important indication for the diagnosis of thoracic outlet syndrome. Lidocaine injection is commonly used in clinical practice in SMB, although there have been some cases of misdiagnosis. Botulinum toxin A (BTX-A) is one of the therapeutic agents in SMB, but whether it is also indicated for SMB diagnosis is controversial. To evaluate the muscle block efficiency of these two drugs, the contraction strength was repeatedly recorded on tibialis anterior muscle in rats. It was found that at a safe dosage, 2% lidocaine performed best at 40 μL, but it still exhibits an unsatisfactory partial blocking efficiency. Moreover, neither lidocaine injection in combination with epinephrine or dexamethasone, nor multiple locations injection could improve the blocking efficiency. On the other hand, injections of 3 U/kg, 6 U/kg, and 12 U/kg BTX-A all showed almost complete muscle block. Gait analysis showed that antagonistic gastrocnemius muscle, responsible for heel rising, was paralyzed for non-specific blockage in the 12 U/kg BTX-A group, but not in the 3 U/kg or 6 U/kg BTX-A group. c-SNAP 25 was stained to test the transportation of BTX-A, and was additionally observed in the peripheral muscles in 6 U/kg and 12 U/kg groups. c-SNAP 25, however, was barely detectable in the spinal cord after BTX-A administration. Therefore, our results suggest that low dosage of BTX-A may be a promising option for the diagnostic SMB of thoracic outlet syndrome. Muscle block is important for the diagnosis and treatment of thoracic outlet syndrome and commonly performed with lidocaine. However, misdiagnosis was observed sometimes. Here, we found that intramuscular injection of optimal dosage lidocaine only partially blocked the muscle contraction in rats, whereas low dosage botulinum toxin, barely used in diagnostic block, showed almost complete block without affecting the central nervous system. This study suggests that botulinum toxin might be more suitable for muscle block than lidocaine in clinical practice.