Mitochondrial DNA (mtDNA) analysis is crucial for the diagnosis of mitochondrial disorders, forensic investigations, and basic research. Existing pipelines are complex, expensive, and require specialized personnel. In many cases, including the diagnosis of detrimental single nucleotide variants (SNVs), mtDNA analysis is still carried out using Sanger sequencing. Here, we developed a simple workflow and a publicly available webserver named Mitopore that allows the detection of mtDNA SNVs, indels, and haplogroups. To simplify mtDNA analysis, we tailored our workflow to process noisy long-read sequencing data for mtDNA analysis, focusing on sequence alignment and parameter optimization. We implemented Mitopore with eliBQ (eliminate bad quality reads), an innovative quality enhancement that permits the increase of per-base quality of over 20% for low-quality data. The whole Mitopore workflow and webserver were validated using patient-derived and induced pluripotent stem cells harboring mtDNA mutations. Mitopore streamlines mtDNA analysis as an easy-to-use fast, reliable, and cost-effective analysis method for both long- and short-read sequencing data. This significantly enhances the accessibility of mtDNA analysis and reduces the cost per sample, contributing to the progress of mtDNA-related research and diagnosis. Display omitted Rossi and colleagues describe a streamlined approach for the analysis of mtDNA using noisy long-read sequencing data. They introduce Mitopore, a user-friendly webserver for efficient, cost-effective mtDNA analysis of SNVs, indels, and haplogroups. The economic efficiency of Mitopore holds great promise to advance research and clinical mtDNA diagnosis.