NAF is a breast fluid that is closely related to the tumor microenvironment and a valuable sample for studying breast cancer. To performan in-depth proteomic analysis of this sample, aliquots of a single NAF digest were analyzed by the following peptide-centric fractionation strategies: a) 30-cm reversed-phase (RP) column on-line with an LTQ-Orbitrap XL; b) off-line strong cation-exchange (SCX) column; and c) pI-based OFFGEL fractionation. All fractions from approaches (b) and (c) were further analyzed on a 10-cm RP column hyphenated to the same mass spectrometer. The RP-30 cm, SCX/RP-10 cm, and OFFGEL/RP-10 cm approaches identified 1676, 2930, and 3240 peptides, which corresponded to 193, 390 and 528 proteins, respectively. In our cumulative dataset, 4466 distinct NAF peptides corresponded to a total of 557 proteins, of which only 34% were identified by all three approaches. No exclusive protein identification was associated to the RP-30 cm approach, while SCX/RP-10 cm and OFFGEL/ RP-10 cm contributed to 28 and 166 exclusive identifications, respectively. Each approach provided additional information related to energy metabolism (fermentation process/ carbohydrate biosynthesis). In conclusion, the pre-fractionation platforms used were complementary for the comprehensive characterization of NAF and our work provides methodological information for future quantitative cancer-related NAF sample studies.