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Leishmaniasis, a group of neglected infectious diseases, encompasses a serious health concern, particularly with visceral leishmaniasis exhibiting potentially fatal outcomes. Nucleoside hydrolase (NH) has a fundamental role in the purine salvage pathway, crucial for Leishmania donovani survival, and presents a promising target for developing new drugs for visceral leishmaniasis treatment. In this study, LdNH was immobilized into fused silica capillaries, resulting in immobilized enzyme reactors (IMERs). The LdNH-IMER activity was monitored on-flow in a multidimensional liquid chromatography system, with the IMER in the first dimension. A C18 analytical column in the second dimension furnished the rapid separation of the substrate (inosine) and product (hypoxanthine), enabling direct enzyme activity monitoring through product quantification. LdNH-IMER exhibited high stability and was characterized by determining the Michaelis-Menten constant. A known inhibitor (1-(β-d-Ribofuranosyl)-4-quinolone derivative) was used as a model to validate the established method in inhibitor recognition. Screening of three additional derivatives of 1-(β-d-Ribofuranosyl)-4-quinolone led to the discovery of novel inhibitors, with compound 2a exhibiting superior inhibitory activity (K i  = 23.37 ± 3.64 µmol/L) compared to the employed model inhibitor. Docking and Molecular Dynamics studies provided crucial insights into inhibitor interactions at the enzyme active site, offering valuable information for developing new LdNH inhibitors. Therefore, this study presents a novel screening assay and contributes to the development of potent LdNH inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)