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Rational use of medicine (RUM) for all medical conditions is crucial in attaining quality of healthcare and medical care for patients and the community as a whole. However, the actual medicine use pattern is not consistent with that of the World Health Organization (WHO) guideline and is often irrational in many healthcare setting, particularly in developing countries. Thus, the aim of the study was to evaluate rational medicine use based on WHO/International Network of Rational Use of Drugs (INRUD) core drug use indicators in Eritrean National and Regional Referral hospitals. A descriptive and cross-sectional approach was used to conduct the study. A sample of 4800 (600 from each hospital) outpatient prescriptions from all disciplines were systematically reviewed to assess the prescribing indicators. A total of 1600 (200 from each hospital) randomly selected patients were observed for patient indicators and all pharmacy personnel were interviewed to obtain the required information for facility-specific indicators. Data were collected using retrospective and prospective structured observational checklist between September and January, 2018. Descriptive statistics, Welch's robust test of means and Duncan's post hoc test were performed using IBM SPSS (version 22). The average number of medicines per prescription was 1.78 (SD = 0.79). Prescriptions that contained antibiotic and injectable were 54.50% and 6.60%, respectively. Besides, the percentage of medicines prescribed by generic name and from an essential medicine list (EML) was 98.86% and 94.73%, respectively. The overall average consultation and dispensing time were 5.46 minutes (SD = 3.86) and 36.49 seconds (SD = 46.83), respectively. Moreover, 87.32% of the prescribed medicines were actually dispensed. Only 68.24% of prescriptions were adequately labelled and 78.85% patients knew about the dosage of the medicine(s) in their prescriptions. More than half (66.7%) of the key medicines were available in stock. All the hospitals used the national medicine list but none of them had their own medicine list or guideline. In conclusion, majority of WHO stated core drug use indicators were not fulfilled by the eight hospitals. The results of this study suggest that a mix of policies needs to be implemented to make medicines more accessible and used in a more rational way. [ABSTRACT FROM AUTHOR]