Whey protein isolate (WPI) is a value-added protein with multiple ingredient applications. A bland flavor is expected in WPI, and off-flavors can limit its use in foods. Recently, a cabbage off-flavor was noted in some WPI. The objective of this study was to characterize the source of cabbage flavor in WPI. WPI with and without cabbage flavor were collected, and descriptive sensory analysis was conducted on the rehydrated WPI using a trained panel and a previously identified sensory language. Volatile compounds were extracted by solvent extraction followed by solvent-assisted flavor evaporation (SAFE), followed by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-olfactometry (GCO), to identify and characterize aroma-active compounds. Dimethyl trisulfide (DMTS) (cabbage aroma) was identified by GCO and GC-MS in WPI with the cabbage flavor. DMTS was quantified by solid-phase microextraction (SPME) with GC-MS. Orthonasal thresholds of DMTS in deodorized water and WPI were determined by ascending forced choice analysis, and descriptive analysis of model systems was used to confirm instrumental results. DMTS levels were 1.94 ± 0.26 and 3.25 ± 0.61 parts per billion (ppb) in WPI with cabbage flavor, and 0.44 ± 0.25 and 0.43 ± 0.18 ppb in those without cabbage flavor. The orthonasal thresholds for DMTS in water and WPI were 0.07 ± 1.28 parts per trillion (ppt) and 0.80 ± 0.45 ppb, respectively. Descriptive analysis of model systems confirmed the role of DMTS in the cabbage off-flavor. Knowledge of the source of this flavor will aid in identification of ways to minimize or prevent DMTS formation in WPI.