<p><i>Leishmania</i> and HIV coinfection is a major public health problem in more than 35 countries worldwide. <i>Leishmania</i> infection is frequently reactivated in VL and HIV coinfected patients. One of the major challenges in the management of VL/HIV coinfection is the lack of an effective drug therapy that not only resolves the first episode of VL but also prevents relapse. This study aimed to assess the in vitro drug susceptibility profile of clinical isolates of <i>Leishmania</i> in HIV positive and negative patients obtained during the first VL episode before treatment and during VL relapses on patients who attended LRTC of University of Gondar, Gondar, northwest Ethiopia, between January 1, 2020, and December 30, 2020. We evaluated the in vitro susceptibility to amphotericin B (AmB), paromomycin (PMM), and miltefosine (MLT) of 30 <i>L. donovani</i> isolates obtained from primary and relapse VL patients with and without HIV co-infection. All 30 strains of <i>L. donovani</i> were tested at the promastigote stage whereas, 10 randomly selected strains of <i>L. donovani</i> were re-tested at the amastigote stage. The mean (± SD) IC₅₀ of AmB, PMM, and MLT tested against promastigotes was 0.221 ± 0.108 µM, 13.49 ± 6.92 µM, 3.77 ± 1.77 µM respectively. Similarly, the mean (± SD) IC₅₀ of AmB, PMM, and MLT against amastigotes was 0.171 + 0.027µM, 10.80 + 4.12 µM, and 3.63 + 1.72 µM, respectively. When the data was disaggregated between primary and relapse cases of VL, the IC₅₀ observed against strains from relapse VL was higher than the IC₅₀ observed against strains from primary VL. The difference in the IC₅₀ against strains from relapse VL with primary VL, was statistically significant (<i>p</i> = 0.03) for AmB against amastigote stages, but not promastigote stage, whereas the difference in IC₅₀ obtained for PMM and MLT against both stages of the parasites was not statistically significant. Likewise, strains from HIV coinfected VL patients with multiple relapses showed an increase in IC₅₀ value for AmB and MLT in both parasite stages. The current study showed that in vitro susceptibility of strains decreased progressively in relapsing patients compared with primary VL patients. Our findings suggest that AmB and MLT resistant parasites may indeed emerge in repeatedly treated relapse VL cases in HIV coinfected patients, warranting the need for continuous monitoring of <i>L. donovani</i> susceptibility to current treatments; and also, the results of such studies reveal the need to re-examine the current therapy policies in HIV coinfected VL patients.</p>