BACKGROUND: There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. METHODS: Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. RESULTS: The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. CONCLUSIONS: We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials.