PURPOSE: The aim of the present study was to establish a SYBR Green-based real-time PCR assay for detection of the Nc5 segment from the Neospora caninum genome. METHODS: The oligonucleotides sequences targeting the Nc5 gene previously reported and designed in-house were validated. Two Primer sets were evaluated and tested in four different combinations. The NP7/NP10 assay was selected and reaction conditions optimized. Efficiency, analytical sensitivity, precision and specificity were assessed. The assay was evaluated in triplicate, in three independent PCR runs performed by two technicians to generate robust results. RESULTS: The standard curve determined by tenfold serial dilutions (1 to 1 × 10- CONCLUSION: The protocol had high specificity, confirmed by melting curve analysis and no cross-reactions with other tested microorganisms. The SYBR Green-based PCR protocol standardized in this study is a highly sensitive and specific method, reproducible and applicable for the detection of N. caninum in different biological samples.