Laboratory diagnosis of leishmaniasis is based on culture, microscopic examination, serological and molecular methods. The gold standard method is to see amastigotes in microscopic examination and to grow promastigotes in Novy, MacNeal, Nicolle (NNN) medium. NNN medium is frequently used for culture all over the world. In our study, it was aimed to investigate whether the use of RPMI-1640 medium is an appropriate method in cases where the gold standard NNN medium is not available for the diagnosis of cutaneous leishmaniasis (CL). Smears were prepared from the needle aspiration fluid sample from the patient who applied to Manisa Celal Bayar University Faculty of Medicine and had lesions suspicious of CL, and were stained with Giemsa for the presence of amastigotes. The samples taken were directly inoculated into RPMI-1640 broth and incubated at 26 °C for the presence of promastigotes. On consecutive days after incubation, it was checked for promastigote growth. Genotyping of the grown isolate was performed with primers and probes specific to the internal transcribed spacer-1 (ITS-1) gene region with the help of real-time polymerase chain reaction. The amastigote form was observed in the microscopic examination of the needle aspiration fluid sample smear preparations taken from the patient. On the other hand, promastigote growth was observed in RPMI-1640 broth from the 3rd day. In addition, the isolate obtained from the CL patient was determined to be Leishmania tropica as a result of the species determination made by genotyping. It is thought that this study is important in terms of suggesting an alternative medium for the diagnosis of leishmaniasis in laboratories where the gold standard NNN medium is easily accessible. RPMI-1640 medium, which is easily obtained and prepared in parasitology laboratories, can help in the diagnosis of the disease and treatment follow-up, Leishmania spp. isolation and drug resistance studies.