Background: Hamstring shortness (HS) contributes to the limit of hip and knee range of motion and can limit the execution of multi-segmental movement exercises such as squatting, thereby altering the muscle activation. The double leg squat (DS) is fundamental exercise for knee injury rehabilitation programs. However, it may aggravate knee joint injury and cause low back pain. The Bulgarian split squat (BSS) is a one-leg squat exercise with greater knee joint stability than DS. Measuring muscle activation during the squat will provide insight into the contribution of hamstring activation and quadriceps and hamstring (Q:H) ratio to the knee in participants with HS. Objects: This study compares hamstring activation and Q:H ratios during squat exercises in individuals with and without HS. Methods: This study classified 30 healthy participants, based on active knee extension, into two groups: with HS and without HS. Surface electromyography was performed to determine muscle activation of the medial hamstring (MH) and biceps femoris of the hamstrings and coactivation of the Q:H. All participants performed DS and BSS. Results: MH activity was significantly higher in the with HS group than in the without HS group during all exercises, and the Q:H ratio was significantly closer to 1.0 during BBS compared to DS. Additionally, the with HS group had a significantly lower Q:H ratio than without HS group. Conclusion: In comparison between exercises, BBS can bring the Q:H ratio closer to 1.0 compared to DS, which may enhance knee stability. However, in between-group comparisons, the increased tension due to the shortening of the hamstrings leads to heightened hamstring activation, resulting in a Q:H ratio that is closer to 1.0 compared to the without HS group. Therefore, relying solely on the Q:H ratio to assess knee stability may be risky.