Leptospirosis diagnosis by MAT requires antibody levels that are typically present only after the first week of symptoms, many days after infection. To improve testing capacity and to develop a fast and reliable solution for the diagnosis of this disease in the first few days after clinical manifestations, the National Reference Laboratory for Leptospirosis/WHO Collaborating Center in Brazil implemented a duplex molecular method by qPCR for human samples for the detection of the gene lipL32, conserved in pathogenic Leptospira spp. In this paper, we describe the overall performance of this protocol in the first 3 months as a standard routine. Detection of pathogenic Leptospira spp. DNA was similar between blood, plasma, and tissue samples, with a limit of detection as low as one cell per sample, and among 391 samples from suspected cases, 174 (44.6%) were positive. The average RNASEP1 control gene detection cycle thresholds (Ct) were 28.4 and 29.8 for positive and negative samples, respectively. The median sample collection interval from the beginning of symptoms was 3 days for positive and 4 days for negative samples, respectively. Neither age, sex, nor the time intervals between sample collection and DNA extraction significantly influenced the results. Surprisingly, positivity was related to the time between DNA extraction and the qPCR reaction. These data support the use of this routine as a diagnostic approach to strengthen the molecular detection of leptospirosis and to develop new strategies.