Binding Fe-peptides is an alternative to increase the bioavailability of iron salts. In this study, iron-binding peptides from whey protein isolate (WPI) were isolated and sequenced by mass spectrometry (MS/MS). WPI was hydrolyzed with pancreatin and the hydrolysate (HWPI) was ultrafiltered (cut off 5 kDa), and fractions HWPI < 5 kDa and HWPI > 5 kDa were lyophilized. The isolation of peptides was carried out by IMAC-Fe3+, in a IDA-Sepharose 6B® column incubated with 200 mmol/L FeCl3. The system was equilibrated with acetic acid buffer (50 mmol/L pH 5.5), with 0.1 mol/L NaCl, and the column, loaded with 2 mL of hydrolysate (50 mg protein/mL). The wash volume was discarded and the bound peptides were eluted with 20 mmol/L Na2HPO4 and monitored at 280 nm. This fraction was lyophilized, desalted in a Sep-Pack column with acetonitrile and formic acid, and sequenced by MS/MS, with ionization by electrospray and Q-TOF analyser. The content of iron-binding peptides was 70% in HWPI > 5 kDa and 50% in HWPI < 5 kDa. The sequencing by MS/MS showed that 82% of identified fragments came from β-lactoglobulin, being YVEELKPTPEGDLEIL and RTPEVDDEALEK the main identified sequences. All fragments showed Glu and/or Asp in their sequences, which carboxylic groups are among the main iron-binding sites. The results suggest that WPI hydrolysis with pancreatin yields peptides with high iron-binding ability. These peptides may form iron-peptide complexes, which might play an important role for obtaining products with high iron bioavailability.