Patellofemoral pain syndrome (PFPS) is one of the most prevalent musculoskeletal conditions of the lower limb. The muscle imbalance between the vastus medialis oblique (VMO) and vastus lateralis (VL) muscles is one of the main factors leading to the development of PFPS. The disparity in research and the necessity to add to the existing literature base led to the development of this study. The aim of this study was to investigate the effect of 2 closed kinetic chain exercises and 1 open kinetic chain exercise on VMO and VL muscle activity. Twenty-two healthy asymptomatic individuals participated in this study. The surface electromyography (EMG) of VMO and VL was measured and used to calculate the VMO:VL ratio during 3 different quadriceps-strengthening exercises (a double leg squat with isometric hip adduction exercise, an open kinetic chain knee extension exercise, and a lunge exercise). The double leg squat with isometric hip adduction exercise was shown to produce a significantly greater VMO:VL ratio (1.14:1) than the other 2 exercises (p = 0.015 and p = 0.005). The open kinetic chain knee extension exercises produced significantly greater activation of VL than the lunge exercise (p = 0.001 and p = 0.036). The lunge exercise produced the VMO:VL ratio (1.18:1) closest to the idealized ratio of 1:1. Potential clinical recommendations can be made proposing the lunge exercise as a key tool in early rehabilitation when restoring preferential VMO:VL ratio is essential. The double leg squat with isometric hip adduction exercise would be useful in maintaining correct patella tracking and selectively strengthening VMO.