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Introduction: The treatment response of multi-drug resistance tuberculosis (MDR-Tuberculosis) patients is mainly dictated by the sputum culture conversion. An earlier culture conversion is a remarkable indicator of the improvement in the treatment response. In this study, we aimed to determine the time to culture conversion and its associated factors among MDR-Tuberculosis patients in All Africa Leprosy, Tuberculosis and Rehabilitation Training Center (ALERT) Hospital, Addis Ababa, Ethiopia. Methods: A retrospective cohort study was conducted on 120 MDR-Tuberculosis patients attending ALERT Hospital from 2018–2022. Kaplan-Meier methods were used to determine the time to initial sputum culture conversion. All relevant laboratory, socio-demographic characteristics, and other clinical data were collected by chart abstraction using a structure data extraction form. The log-rank test was used to determine the survival rate. To identify the predictors of culture conversion, bivariate and multivariate Cox proportional hazard regression analysis was used. The hazard ratio (HR) with a 95% confidence interval was used to estimate the effect of each variable on the initial culture conversion. A test with a P value of < 0.05 was considered statistically significant. Results: From the total of 120 study participants, 89.2% (107/120) have shown a successful culture conversion. The median age of the participants was 30 years (IQR = 12). The study participants were followed for 408.6 person-months (34.05 person-years). The median time to initial sputum culture conversion was 80 days. The median time to initial sputum culture conversion among HIV-positive and HIV-negative participants was 61 days (IQR = 58–63.5) and 88 days (IQR = 75–91), respectively. HIV-negative and patients with previous treatment history were shown to be the predictor for a prolonged time to initial sputum culture conversion, (aHR = 0.24 (95% CI: 0.1–0.4), P value <0.001) and (aHR = 0.47 (95% CI: 0.31–0.71), P value <0.001) respectively. Conclusion: The median time to sputum culture conversion for HIV positive was found to be 61 days in our study. Notably, patients with a history of previous anti-tuberculosis treatment, HIV-negative status, and higher bacillary load at baseline exhibited delayed culture conversion. These findings underscore the importance of considering such patient characteristics in the management of MDR-TB cases, as tailored interventions and close monitoring may lead to more favorable treatment outcomes. By identifying individuals with these risk factors early in the treatment process, healthcare providers can implement targeted strategies to optimize patient care and improve overall treatment success rates in MDR-TB management programs. [ABSTRACT FROM AUTHOR]