Previous studies for treating Patellofemoral Pain Syndrome (PFPS) are controversial regarding the effectiveness in alleviating anterior knee pain (AKP). The muscular imbalance between the vastus medialis oblique/vastus lateralis (VMO/VL) may be the underlying mechanical issue causing PFPS. It is hypothesized that Botox can decrease the force production capability of the lateral musculature mechanically similar to surgery. Strengthening the VMO while using Botox treatment can alleviating the muscular imbalance that occurs with subjects with PFPS. A double blind study, having all participants blinded and uninformed of the injection contents, was implemented to test this hypothesis testing three female subjects. Four knees were treated. One subject received the Botox treatment and serially a placebo injection in the other limb. Two other subjects received placebo injections. EMG was executed to evaluate functional testing and the performance of the injections during extension exercises. Electromyography (EMG) data were collected from the muscle groups while the subjects performed forceful knee extension activities on an isokinetic dynamometer. In addition, kinetic jump data and self-reports of pain and activity were collected. Data were collected four times during a 12-week period. The subject who received Botox injections expressed a significant decrease in reported PFP and an increase in daily activities. Botox was safe and effective in eliminating anterior knee pain. The VMO and VL resulted in similar fatigue indices at the completion of the 12- week study. The VMO and VL both resisted fatigue during at week 12.