Plasmodium vivaxis the second most common malaria parasite in Ethiopia. It has been treated with chloroquine (CQ) for the past seven decades. However, the emergence of CQ-resistant strains in the nation urged the Federal Ministry of Health of Ethiopia to review its national malaria treatment guideline in 2018.In the revised guideline, thefirst-line treatment for uncomplicatedP. vivaxinfection is a combination of CQ and primaquine (PQ). Thus, the present study was designed to evaluate the in vivo efficacy of CQ and PQ combination therapy against clinicalP. vivaxmono-infection in one of the malaria-endemic areas of Ethiopia. Anopen-label prospective clinical trialwas conducted in the Limmu Kossa District, Jimma zone, Southwest Ethiopia, from September 2023 to March 2024. A total of 108 patients were recruited for the study. All participants received treatment with CQ at a dosage of 25mg/kg over three days, followed by PQ at 0.25mg/kg for 14 consecutive days. Patients were monitored for 42days for any signs of treatment failure and malaria clinical symptoms, as per the World Health Organization (WHO) guidelines for anti-malarial drug evaluation. Additionally, haemoglobin (Hb) levels, body temperature,any adverse events, and signs of haemolysis were assessed.Data was analysed using R-software (version 4.0.0) and a significant level was considered at p < 0.05. The median age of the patients was 23years, ranging from 2.5 to 62years. Of the 108 patients initially recruited, 100 completed the 42-day follow-up period. The combination therapy of CQ and PQ for uncomplicated clinicalP. vivaxmalaria demonstrated excellent therapeutic efficacy, with a 100% cure rate observed at both day 28 and day 42. Additionally, the recommended low dose of PQ (0.25mg/kg) was well-tolerated, with no signs of. Additionally, most common malaria symptoms were disappeared early in the follow-up period. The combination of CQ plus PQ has exhibited excellent efficacy against uncomplicatedP. vivaxmalaria mono-infections. To preserve this efficacy, it is critical to ensure patients adhere to the full course of PQ treatment, despite its extended duration. Therefore, health authorities should put emphasis on the boosting of the public on the importance of finishing the prescribed medication regimen.