Background: Acute kidney injury (AKI) is one of the most common causes of neonatal morbidity and mortality. Diagnosing AKI in neonates is challenging as it lacks specific signs, symptoms, and biomarkers. However, detecting AKI in critically ill neonates is crucial to determine appropriate management and prevent complications. Cystatin C (CysC) has been recognized as a superior kidney biomarker reflecting kidney function in neonates. The objective of this study is to evaluate the diagnostic value of CysC as an AKI biomarker in critically ill neonates. Methods: We performed a diagnostic test between cystatin C-based estimated glomerular filtration rate (eGFR-CysC) and serum creatinine-based estimated glomerular filtration rate (eGFR-SCr) as the gold standard to diagnose AKI in 135 critically ill neonates treated in Cipto Mangunkusumo National Hospital from July 2017 to January 2018. Results: Prevalence of AKI was 23.7% predominantly in neonates with a very preterm gestational age, low birthweight, probable sepsis, and those receiving invasive oxygen therapy or nephrotoxic drugs. The proportion of AKI based on neonate RIFLE criteria was 72.7% risk, 18.9% injury, and 9% failure. eGFR-CysC had the following parameters: sensitivity, 84.8%; specificity, 61.8%; PPV, 41.8%; NPV, 89.7%; LR(+), 2.2; LR(−), 0.24; and accuracy, 67.4%. The AUROC for CysC was 84.9%. The optimal cut-off value for CysC was 1.605 mg/l. Conclusions: CysC may be used as a screening biomarker of AKI in critically ill neonates; yet, it was not superior to serum creatinine. [Figure not available: see fulltext.]. © 2020, IPNA.