Various hair follicle (HF)-associated disorders, such as acne vulgaris, hidradenitis suppurativa, and alopecia areata, are linked to dysbiosis, an imbalance between resident and pathogenic microbes. Characterization of the HF and skin microbiome employs techniques such as 16S rRNA gene sequencing and metagenomic shotgun sequencing, with the latter providing comprehensive taxonomic and functional insights. However, relic DNA from dead microbes and free environmental DNA can persist in samples, meaning that metagenomic data does not exclusively reflect living microbiota. For functional studies on HF dysbiosis or to assess potential therapeutic interventions, we describe here how propidium monoazide (PMA) treatment can be performed before (metagenomics) sequencing to distinguish viable microbial communities. Furthermore, we exemplify qPCR and (fluorescent) in situ hybridization (ISH) of two alternative viability screening methods for the HF and scalp microbiome.