Background: Malaria is a severe disease, so delay in treatment could increase morbidity and mortality. World Health Organization recommends confirmation of the diagnosis by laboratory test before initiation of therapy. Smear microscopy and polymerase chain reaction are methods approved by WHO for diagnosing malaria, but they are time-consuming, operator-dependent, and require laboratory staff training. WHO has recommended the Blood-based Rapid Diagnostic Test (RDT) as an acceptable method for diagnosing malaria. It provides rapid results and can be performed with limited resources. Methods: Four hundred and fourteen febrile cases admitted to Khartoum North General Hospital, Khartoum, Sudan during January 2021 - June 2021 with clinical suspicion of malaria were examined for the presence of asexual forms of Plasmodium falciparum in peripheral blood smears. Blood smear microscopy-positive patients who matched the acceptance criteria were included, while negative patients were selected as control cases. Blood and urine samples were examined with the same RDT kits designed for blood. Results: Fifty-eight blood smear-positive cases and 50 febrile blood smear-negative cases were enrolled in the study. The sensitivity and specificity of urine-based RDT were 82.76% and 92.00%, respectively, while blood-based RDT was 96.55% and 96.00%. Conclusion: This study aimed to evaluate the performance of urine-based RDT through comparison with blood-based RDT and the use of blood smear microscopy as the reference method. Although the results showed acceptable performance of both tests, more extensive sample size studies should be conducted to consider urine samples as an alternative sample for diagnosing Plasmodium falciparum malaria.