The masseter muscle, a key orofacial muscle, demonstrates unique anatomical and functional properties, including sexual dimorphism in myosin heavy chain (MyHC) expression and complex fiber architecture. Despite its importance in mastication and relevance to various disorders, phenotypic characterization of the masseter remains limited. Conventional fluorescence microscopy has been a cornerstone in muscle fiber typing, reliably identifying MyHC isoforms and measuring fiber cross-sectional areas. Building on this foundation, confocal microscopy offers complementary advantages, such as enhanced resolution, increased flexibility for multiplexing, and the ability to visualize complex structures in three dimensions. This study presents a detailed protocol for using confocal microscopy to achieve high-resolution imaging and molecular characterization of masseter muscle cryosections. By leveraging advanced technologies such as white light lasers and extended z-length imaging, this method ensures precise spectral separation, simultaneous multichannel fluorescence detection, and the ability to capture muscle architecture in three dimensions. The protocol includes tissue preparation, immunostaining for MyHC isoforms, and postprocessing for fiber segmentation and quantification. The imaging setup was optimized for minimizing signal bleed through, improving the signal-to-noise ratio, and enabling detailed visualization of muscle fibers and molecular markers. Image postprocessing allows for quantification of the cross-sectional area of individual fibers, nuclei location measurements, and identification of MyHC isoforms within each fiber. This confocal microscopy–based protocol provides similar resolution and contrast compared to conventional techniques, enabling robust multiplexed imaging and 3D reconstruction of muscle structures. These advantages make it a valuable tool for studying complex muscle architecture, offering broad applications in muscle physiology and pathology research.