Animal models clearly illustrate that the maintenance of skeletal muscle mass depends on the function and interaction of a heterogeneous population of resident and infiltrating mononuclear cells. Several lines of evidence suggest that mononuclear cells also play a role in muscle wasting in humans, and targeting these cells may open new treatment options for intervention or prevention in sarcopenia. Methodological and ethical constraints have perturbed exploration of the cellular characteristics and function of mononuclear cells in human skeletal muscle. Thus, investigations of cellular phenotypes often depend on immunohistochemical analysis of small tissue samples obtained by needle biopsies, which do not match the deep phenotyping of mononuclear cells obtained from animal models. Here, we have developed a protocol for fluorescence-activated cell sorting (FACS), based on single-cell RNA-sequencing data, for quantifying and characterizing mononuclear cell populations in human skeletal muscle. Muscle stem cells, fibro-adipogenic progenitors, and two subsets of macrophages (CD11c^+/–) are present in needle biopsies in comparable quantities per milligram tissue to open surgical biopsies. We find that direct cell isolation is preferable due to a substantial shift in transcriptome when using preculture before the FACS procedure. Finally, in vitro validation of the cellular phenotype of muscle stem cells, fibro-adipogenic progenitors, and macrophages confirms population-specific traits. This study demonstrates that mononuclear cell populations can be quantified and subsequently analyzed from needle biopsy material and opens the perspective for future clinical studies of cellular mechanisms in muscle wasting.