BACKGROUND: Intestinal parasitic diseases affect millions of people worldwide. Numerous commercial molecular methods detecting digestive parasites have been developed recently, including multiplex PCR assays able to identify multiple parasites at once. Several studies have demonstrated that the efficacy of these molecular methods is dependent on the specific protocols employed at each stage of the process including pretreatment, extraction and amplification. However, previous studies have exclusively focused on one of these steps, without considering the others. The objective of the present study was to evaluate the performances of molecular tools for Cryptosporidium parvum detection in stool samples, considering all steps of the process simultaneously. METHODS: 30 distinct combinations of protocols were evaluated corresponding to three pre-treatment methods, four DNA extraction techniques and six DNA amplification assays. The performances of these combinations were evaluated in terms of detection limit. RESULTS: We showed that different combinations yielded varying results. The FTD® Stool Parasite technique proved to be the most effective, achieving 100 % detection. Manual extraction methods demonstrated excellent outcomes, although they are time-consuming. The optimal approach for detecting C. parvum DNA is a combination of mechanical pretreatment, the Nuclisens® Easymag® extraction method, and the FTD® Stool Parasite DNA amplification method. CONCLUSION: This work shows that the molecular diagnosis should consider all stages. A PCR method may not be effective with an unsuitable extraction technique, but can yield optimal results with an appropriate one.