Background: During the first postnatal weeks, infants have abrupt changes in fluid weight that alter serum creatinine (SCr) concentration, and possibly, the evaluation for acute kidney injury (AKI). Methods: We performed a prospective study on 122 premature infants to determine how fluid adjustment (FA) to SCr alters the incidence of AKI, demographics, outcomes, and performance of candidate urine biomarkers. FA-SCr values were estimated using changes in total body water (TBW) from birth; FA-SCR = SCr × [TBW + (current wt. – BW)]/ TBW; where TBW = 0.8 × wt in kg). SCr-AKI and FA-SCr AKI were defined if values increased by ≥ 0.3 mg/dl from previous lowest value. Results: AKI incidence was lower using the FA-SCr vs. SCr definition [(23/122 (18.8 %) vs. (34/122 (27.9 %); p < 0.05)], with concordance in 105/122 (86 %) and discordance in 17/122 (14 %). Discordant subjects tended to have similar demographics and outcomes to those who were negative by both definitions. Candidate urine AKI biomarkers performed better under the FA-SCr than SCr definition, especially on day 4 and days 12–14. Conclusions: Adjusting SCr for acute change in fluid weight may help differentiate SCr rise from true change in renal function from acute concentration due to abrupt weight change.