Background: DMD is a muscle-wasting disorder without a cure caused by disruptive mutations in the DMD gene. The mdx mouse model is commonly used in DMD studies but it has a subtle phenotype compared to wild type, making it a challenging model to assess muscle strength. Therefore, methods to assess its phenotype are necessary. Here, we show that the acoustic startle reflex can be an accurate test to differentiate between mdx-5cv and c57/black (WT) mice. Methods: We assessed the startle reflex of 6 month-old mdx-5cv (n = 20) and c57/black (WT, n = 19) mice to a range of acoustic stimuli. Mice were placed into individual cubicles that contained an isolation chamber capable of holding them still on top of a force transducer. Mice were exposed to a random sequence of tones varying from 20 to 115 dB with intervals ranging from 10 to 20 seconds. Startle responses were recorded for 150 milliseconds from the sound onset and sampled every millisecond. The outcomes per tone are maximum force, average force, and time of maximum force. Data is shown as average ± SD. Results: WT mice developed on average ~55% higher maximum force than mdx-5cv (0.491 ± 0.167 N vs. 0.316 ± 0.137 N, respectively). WT mice developed on average ~81% higher average force than mdx-5cv (0.049 ± 0.016 N vs. 0.027 ± 0.007 N, respectively). Finally, the median average time to reach maximum force was found to be ~20% longer in the mdx-5cv mice. Conclusion: The startle reflex test can statistically differentiate the phenotype of mdx-5cv and WT mice models. This holds potential as a novel method for assessing functional gains in DMD research, providing valuable insights into the effectiveness of new treatments aimed at restoring dystrophin expression in DMD patients.