Many skeletal muscle proteins are present in a cell-specific or fibre-type dependent manner. Stimuli such as exercise, aging, and disease have been reported to result in fibre-specific responses in protein abundances. Thus, fibre-type-specific determination of the content of specific proteins provides enhanced mechanistic understanding of muscle physiology and biochemistry compared with typically performed whole-muscle homogenate analyses. This analysis, however, is laborious and typically not performed. We present a novel dot blotting method for easy and rapid determination of skeletal muscle fibre type based on myosin heavy chain (MHC) isoform presence. Requiring only small amounts of starting muscle tissue (i.e., 2-10 mg wet weight), muscle fibre type is determined in one-tenth of a 1-3-mm fibre segment, with the remainder of each segment pooled with fibre segments of the same type (I or II) for subsequent protein quantification by western blotting. This method, which we validated using standard western blotting, is much simpler and cheaper than previous methods and is adaptable for laboratories routinely performing biochemical analyses. Use of dot blotting for fibre typing will facilitate investigations of fibre-specific responses to diverse stimuli, which will advance our understanding of skeletal muscle physiology and biochemistry.