Real-time PCR (qPCR) has emerged as a promising alternative to be used in the diagnosis of leishmaniasis. However, a consensus on the standardized approach for this purpose is yet to be established. This systematic review aims to highlight relevant aspects for the development of possible consensus protocols. The study included publications from January 2011 to July 2023 using different databases. Studies focusing on qPCR standardization for the diagnosis of leishmaniasis were included. The collected data encompassed study group details, geographical locations, sample types and quantities, molecular targets, and qPCR methodologies, which were subsequently pooled and analyzed. Forty-three studies conducted in diverse regions of the world were included. The majority were visceral leishmaniasis, followed by cutaneous leishmaniasis and post-kala-azar dermal leishmaniasis. Overall, invasive samples demonstrated higher sensitivity (> 90%) compared to non-invasive samples (< 90%), but both exhibited high specificity (> 85%). Furthermore, the kDNA minicircle was identified as the most promising molecular target, with a discreet preference for the use of TaqMan probes over SYBR Green. Various qPCR approaches have exhibited promising results in the diagnosis of leishmaniasis. However, further studies exploring non-invasive sampling techniques in conjunction with appropriate molecular targets are essential to establish efficient and reliable diagnostic techniques.