Abstract Mitochondrial DNA (mtDNA) encodes the core subunits for OXPHOS and is essential in eukaryotes. mtDNA is packed into distinct foci (nucleoids) inside mitochondria, and the number of mtDNA differs between cell-types, and is affected in several human diseases. Today, common protocols estimate per-cell mtDNA-molecule numbers by sequencing or qPCR from bulk samples. However, this does not allow insight into cell-to-cell heterogeneity and can mask phenotypical sub-populations. Here, we present mtFociCounter , a single-cell image-analysis tool for reproducible quantification of nucleoids and other foci. mtFociCounter is a light-weight, open-source freeware and overcomes current limitations to reproducible single-cell analysis of mitochondrial foci. We demonstrate its use by analysing 2165 single fibroblasts, and observe a large cell-to-cell heterogeneity in nucleoid numbers. In addition, mtFociCounter quantifies mitochondrial content and our results show good correlation (R=0.90) between nucleoid number and mitochondrial area, and we find nucleoid density is less variable than nucleoid numbers in wild-type cells. Finally, we demonstrate mtFociCounter readily detects differences in foci numbers upon sample-treatment, and applies to superresolution microscopy. Together, we present mtFociCounter as a solution to reproducibly quantify cellular foci in single cells and our results highlight the importance of accounting for cell-to-cell variance and mitochondrial context in mitochondrial nucleoid analysis.