The majority of eukaryotic microbial biodiversity, particularly within certain groups, remains underexplored. Efforts to address this knowledge gap are often limited, as universal methods fail to capture the full scope of microbial diversity. Tailored metabarcoding approaches, in combination with modern techniques, such as the application of high-throughput sequencing, offer a more effective solution. Autotrophic euglenids represent one such group that is frequently overlooked due to their habitat preferences and complex genetic features, causing the inadequacy of universal primers. To address these challenges, we present a customized workflow using group-specific primers targeting the V2 region of the 18S rDNA, optimized biomass harvesting, and refined DNA extraction protocols. This methodology was validated on 190 samples from 16 Polish water bodies, resulting in improved detection and a clearer picture of euglenid diversity and distribution. Our guide provides a comprehensive step-by-step protocol—from sampling to bioinformatics analysis, specifically designed to enhance the accuracy and resolution of euglenid biodiversity assessments.