Background: Neutrophil gelatinase-associated lipocalin (NGAL) has been shown to be a useful marker of kidney injury in people and dogs, but has not been described in horses. Objectives: The aim of the study was to validate a commercially available porcine-specific ELISA to measure serum concentrations of equine NGAL. Methods: Intra- and interassay imprecisions were evaluated by multiple measurements on equine serum pools. Assay inaccuracy was determined by the linearity under dilution. Overlapping performance was assessed by measuring NGAL concentrations in horses with normal and elevated serum creatinine levels. Results: Intra- and interassay imprecision (coefficient of variation) ranged from 5.35% to 28.39%. The ELISA showed no signs of inaccuracy. Overlapping performance was acceptable, as the assay was able to detect the expected differences of NGAL levels in horses with normal and elevated serum creatinine concentrations. Conclusions: Equine serum NGAL concentrations could be quantified reliably using the porcine-specific ELISA.