Background: The most frequent etiology of chronic liver illnesses globally is non-alcoholic fatty liver disease (NAFLD). To identify insulin resistance (IR) in NAFLD cases, the homeostasis model assessment estimate of IR (HOMA-IR) is being studied. Objective: To estimate the role of HOMA-IR in the context of NAFLD diagnosis. Subjects and methods: This retrospective cross-sectional study was conducted during the period from 2020 to 2021 in Benha University Hospitals, Egypt. It was conducted on 205 subjects; 102 NAFLD patients and 103 normal subjects. Entire subjects were assessed by complete history taking and physical examination. Liver function tests and lipid profile, fasting blood glucose (FBG), fasting insulin (FI) level and HOMA-IR were measured. Abdominal ultrasound (US) for diagnosis and grading of fatty liver was done for each subject. Results: In NAFLD patients, the mean age was 45.54 ± 9.56 years, 71.6% were females. Diabetes mellitus, hypertension, weight, and body mass index (BMI) were higher in NAFLD group. NAFLD cases had significantly higher FBG levels compared to those without NAFLD (107.40±5.34 mg/dl vs 85.10±0.71 mg/dl, p=0.047). Patients with NAFLD had significantly higher FI levels in comparison with those without NAFLD (4.62±0.86 μU/ml vs 4.39±0.56 μU/ml, p=0.026). Patients with NAFLD had significantly greater HOMA-IR in comparison with those without NAFLD (1.21±0.06 vs 0.92±0.01, p < 0.001), with best cut off (>1.01), sensitivity (Sn) was 64.71% and specificity (Sp) was 82.52%. HOMA-IR showed ascending level with increased grade of fatty liver. Conclusion: FI, FBG and HOMA-IR are correlated with diagnosis of NAFLD. HOMA-IR is an biomarker of the degree of hepatic steatosis and can be used to identify individuals for further testing.