Introduction: Gentamicin was associated with an apparent increase in the number of neonates with symptoms of hypocalcemia, including seizures. Aim of the study: This study aimed to assess the effect of gentamicin on calcium /creatinine ratio in urine and serum calcium in neonates. Methods: This case-control study included 100 children, divided into two groups. Group I: Fifty neonates received gentamicin. Group II: Fifty neonates did not receive antibiotics at all (control group), Serum calcium and urinary calcium/creatinine ratio, were done at 1st day of admission, and at day 3 and 6 of aminoglycosides therapy. Results: On admission, serum calcium showed a non-significant difference between both groups, Pvalue was 0.453. On the 3rd day, serum calcium was significantly lower in cases compared to controls with P-value was