Bilateral muscular imbalance can increase the risk of injury and negatively impact on sporting performance. Bilateral muscular imbalances are typically calculated as ([side 1 − side 2]/reference value) × 100, to provide a percentage value of the difference between limbs. Using different numerator (right-left or strong-weak) or reference values (left, right, strong, weak, average of the 2) could mask or inflate the true difference value. This study aimed to compare the bilateral muscular imbalance ratio calculations, using the absolute difference between limbs as the numerator and the 5 different options as reference values. Twenty-three males (21.6 ± 1.9 years, 1.80 ± 0.06 m, 80.5 ± 13.8 kg) and 11 females (20.8 ± 1.5 years, 1.62 ± 0.03 m, 68.0 ± 6.5 kg) performed the one-legged 6-m timed test and the one-legged triple-hop distance test. The 5 possible combinations were compared with a 2 (sex) × 2 (functional test) × 5 (calculation method) analysis of variance for each test. Significant differences ( p ≤ 0.05) were found between sex when the right leg was used as the reference value (men: 6.1%, women: 9.1%), and within calculation methods for men (range: 5.9–6.5%) and women (range: 8.4–9.4%), with low effect sizes (range: 0.07–0.26). These findings demonstrate that using a different reference value for calculating bilateral muscular imbalances does not result in a practically significant difference. These findings can be used to inform a more standardized calculation method which will afford conditioning coaches a more correct evaluation and monitoring of training and rehabilitation programs.