Progress in liquid chromatography and mass spectrometry technologies offers a great opportunity for the determination of bioactive peptides. Nevertheless, in many cases, the direct application of this technology does not enable the detection of the investigated peptides due to serious signal suppression. This is the case of lunasin, a cancer preventive, anti-inflammatory, and cholesterol-reducing peptide originally isolated from soybean and later found in some cereals. Most methods applied for the quantitation of this peptide were immunological and based on the detection of just a fragment of the lunasin sequence. At this regard, there is a peptide commercially available with a sequence similar to lunasin but differing in just one amino acid that has been wrongly used for the quantitation of lunasin. The use of high resolution mass spectrometry has enabled to be aware of this issue and of the need for new methods enabling the reliable identification and determination of lunasin. However, when different approaches were evaluated in this work for the reduction of the interferences originating signal suppression, such as matrix dilution, previous lunasin purification by reversed-phase or ion-exchange solid-phase extraction, and use of different chromatographic columns, no one resulted successful in the case of soybean. Just a one-dimensional separation of the soybean extract by isoelectrofocusing followed by a second dimension separation by reversed-phase liquid chromatography enabled a significant reduction of matrix interferences and the detection of lunasin in soybean products by high resolution mass spectrometry with a time of flight (TOF) analyzer. After method optimization, selectivity, linearity, accuracy, precision, and limits of detection and quantitation were evaluated, being possible to quantitate as low as 25ng/mL (1.5μg lunasin/g protein). Concentration of lunasin in the analyzed soybean flour and textured soybean ranged from 14.0 to 22.5mg lunasin/g protein.