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Background: Insulin therapy can be inconvenient, painful, burdensome, and restrict patients' daily activities and health related quality of life (HRQOL) due to improper injection techniques or the nature of administration. Objective: This study aimed to assess insulin injection practice, HRQOL and predictors among patients treated with insulin at Tikur Anbessa Specialized Hospital (TASH). Methods: An institutional-based cross-sectional study was conducted among diabetes patients on insulin therapy from May to June 2022. A structured questionnaire was used to collect patient characteristics and insulin injection practice. The validated Amharic version of an EQ-5D-5L tool was used to assess the HRQOL. The data was analyzed using SPSS version 26. The patient data were summarized using descriptive statistics. One-way ANOVA using Kruskal–Wallis H tests was used to assess factors that predict insulin handling practice scores. Multivariate linear regression analysis was used to assess factors affecting HRQOL among diabetes patients treated with insulin. The EQ5D-5L utility scores of the patients were calculated using disutility coefficients taken from the Ethiopian general population. Statistical significance was declared at p-value < 0.05. Results: Of 319 patients who agreed and completed the survey, 51.1% of them were males. Almost half of the participants (n = 158) were > 50 years of age. Among the study participants, 62.1% were only on intermediate acting insulin. A significantly higher proportion of participants 291(91.2%) in this study were taking insulin two times per day. Most of the participants 234(73.4%) had fair practice with a median insulin handling practice score of 38 out of 56. Patient characteristics such as age, educational status, occupation, disease duration, and type of diabetes were significantly association with insulin injection practice (p < 0.05). The mean ± SD utility score of patients were 0.89 ± 0.19 (ranged from -0.04 to 1). Being female (β = -5.42, 95%CI:-8.63,-2.21, p = 0.001) and treated for type-I diabetes mellitus (β = + 9.04, 95%CI: 4.23,13.85, p-value < 0.0001) were significantly associated with HRQOL of patients on insulin therapy. Conclusion: The study participants had fair practices in insulin handling, storage, and administration techniques, and it was seen that male and type one diabetes patients have a better quality of life compared to their counterparts. [ABSTRACT FROM AUTHOR]