Despite many efforts undertaken to control the human immunodeficiency virus epidemic, it remains to be the major global public health challenge. With expanding access to pediatric antiretroviral therapy, children are more likely to experience treatment failure. All previous studies conducted in Ethiopia estimated treatment failure using only clinical and CD4 criteria. Thus, the ART failure rate is expected to be underestimated in our country.To assess the incidence and predictors of treatment failure among children receiving first-line ART in general hospitals of Mekelle and Southern Zones of Tigray region, Ethiopia, 2019.Retrospective follow up study was employed. The sample size was estimated based on a Log rank test using Stata V-13 and all 404 charts were taken for review. Data were collected by extraction tool; entered using Epi-data manager; cleaned and analyzed by Stata V-14. Data were described using the Kaplan-Meier curve, Log rank test, life table, and crude hazard ratios and analyzed using adjusted hazard ratios and p-value by Cox proportional hazard regression. Any variable at P <0.05 in the bi-variable analysis was taken to multi-variate analysis and significance was declared at P≤ 0.05. Data were presented using tables, charts, and texts.The incidence rate of ART failure was 8.68 (95% CI 7.1 to 10.6) per 1000 person-month observations with a total of 11,061.5 person-month observations. Children who had tuberculosis at baseline [AHR=2.27; 95% CI 1.12-4.57], advanced recent WHO stage [AHR=5.21; 95% CI 2.75-9.88] and sub-optimal ART adherence [AHR=2.84, 95% CI 1.71-4.72] were at higher hazard for first-line treatment failure. Besides this having a long duration of ART follow up [AHR=0.85; 95% CI 0.82-0.87] was found to be protective against treatment failure.The incidence of first-line ART failure was grown as a major public health concern. Treatment failure was predicted by the duration of follow up, advanced recent WHO stage, sub-optimal adherence, as well as the presence of tuberculosis at baseline. Hence, it is better to give priority for strengthening the focused evaluation of the WHO clinical stage and tuberculosis co-infection at baseline with continuous adherence monitoring.