An optimized real-time qPCR assay was chosen for L. infantum DNA detection in urine samples. The chosen of the most adequate and feasible DNA extraction protocol was pivotal. The detection limit reached was 5 fg (~ 0.025 parasites) per μL of urine. Correlations with implementation of treatment were found. The assay indicated a good negative predictive value in urine samples from untreated suspected patients.