The success in development of molecular markers, phylogenetic analysis, and genetic engineering relies heavily on high quality organelle genomes. Any errors in the assembly of these genomes can result in inaccurate downstream analysis. In this article, we present a complete protocol for assessing the assembly quality of organelle genomes using sequencing depth and coverage plot. The protocol consists of nine steps that can be divided into three sections, allowing users to map sequence reads to the assembly, calculate sequence depth and coverage, and plot the data using a custom script, respectively. We provide detailed instructions for setting up the computational environment and running the analytical software tools. This protocol is particularly suitable for users with little bioinformatic experience and will play a vital role in ensuring the high assembly quality of organelle genomes.