Skeletal muscle structural assembly (and its remodeling in response to loading-unloading states) can be investigated macroscopically by assessing muscle architecture, described as fascicle geometric disposition within the muscle. Over recent decades, various medical imaging techniques have been developed to facilitate the in vivo assessment of muscle architecture. However, the main advantages and limitations of these methodologies have been fragmentally discussed. In the present article, the main techniques used for the evaluation of muscle architecture are presented: conventional B-mode ultrasonography, extended-field-of-view ultrasound, 3-D ultrasound and magnetic resonance imaging-based diffusion tensor imaging. By critically discussing potentials and shortcomings of each methodology, we aim to provide readers with an overview of both established and new techniques for the in vivo assessment of muscle architecture. This review may serve as decision guidance facilitating selection of the appropriate technique to be applied in biomedical research or clinical routine.