Quantification of mitochondrial DNA (mtDNA) content is an essential tool for the diagnosis of mtDNA depletion syndrome (MDS). Samples collected and processed for anatomopathology studies represent a unique source of archived biological material. Thus, the possibility to study mtDNA copy number in these specimens would be a useful way to screen for MDS. In this study, we designed and validated the methodology to determine mtDNA content by quantitative real-time polymerase chain reaction (qRT-PCR) in formalin-fixed paraffin-embedded (FFPE) muscle tissue. We studied 14 frozen muscle biopsies and compared the results with a portion of the same biopsy embedded in paraffin. Our results showed a similar variability among frozen and FFPE muscle biopsies. Patients with MDS detected in frozen muscle were also confirmed in their corresponding FFPE samples, which validate the usefulness of this approach. We conclude that the analysis of mtDNA copy number in FFPE muscle tissue by qRT-PCR is a useful method for the molecular screening of patients suspected to have MDS when frozen biopsies are not available. Analysis of these samples would facilitate retrospective studies and diagnostic procedures.