BackgroundNeutrophil gelatinase–associated lipocalin (NGAL) is a protein that is used in human medicine as a real‐time indicator of acute kidney injury (AKI).HypothesisDogs with AKI have significantly higher plasma NGAL concentration and urine NGAL‐to‐creatinine ratio (UNCR) compared with healthy dogs and dogs with chronic kidney disease (CKD).Animals18 healthy control dogs, 17 dogs with CKD, and 48 dogs with AKI.MethodsOver a period of 1 year, all dogs with renal azotemia were prospectively included. Urine and plasma samples were collected during the first 24 hours after presentation or after development of renal azotemia. Plasma and urine NGAL concentrations were measured with a commercially available canine NGAL Elisa Kit (Bioporto® Diagnostic) and UNCR was calculated. A single‐injection plasma inulin clearance was performed in the healthy dogs.ResultsMedian (range) NGAL plasma concentration in healthy dogs, dogs with CKD, and AKI were 10.7 ng/mL (2.5–21.2), 22.0 ng/mL (7.7–62.3), and 48.3 ng/mL (5.7–469.0), respectively. UNCR was 2 × 10−8 (0–46), 1,424 × 10−8 (385–18,347), and 2,366 × 10−8 (36–994,669), respectively. Dogs with renal azotemia had significantly higher NGAL concentrations and UNCR than did healthy dogs (P < .0001 for both). Plasma NGAL concentration was significantly higher in dogs with AKI compared with dogs with CKD (P = .027).Conclusions and Clinical ImportancePlasma NGAL could be helpful to differentiate AKI from CKD in dogs with renal azotemia.