The loaded step-up exercise allows strength and conditioning practitioners to incorporate a unilateral resistance for athletes while performing extension at the hip, knee, and plantar flexion at the ankle. This study evaluated the activation of the biceps femoris (BF), gluteus maximus (GMx), gluteus medius (GMe), rectus femoris, semitendinosus (ST), vastus lateralis, and vastus medialis during 4 variations of the step-up exercise to assess the specific muscle training stimulus of each exercise variation. The exercises included the step-up, crossover step-up, diagonal step-up, and lateral step-up. Fifteen women who regularly engaged in lower body resistance training performed the 4 exercises with 6 repetition maximum loads on a 45.72-cm (18-in.) plyometric box. Data were collected with a telemetered electromyography (EMG) system, and root mean square values were calculated for EMG data for eccentric and concentric phases. Results of a repeated-measures analysis of variance revealed a variety of differences in muscle activation between the exercises (p ≤ 0.05). The results indicated that the crossover step-up elicited the greatest concentric muscle activation for the GMe, whereas the step-up elicited greatest eccentric activation for the GMe and greatest activation for the GMx, BF, and ST in both concentric and eccentric phases. These findings can be used by practitioners to inform exercise selection to best target and maximally activate a variety of hip and thigh musculature.