The present study was designed to identify and compare the infrared absorption spectra of two human breast cancer cell lines: MCF-7 (estrogen receptor expressed, ER+) and SKBr3 (estrogen receptor non-expressed, ER–). Comparison between SKBr3 and MCF-7 cells revealed differences in the following absorption band areas: 1087 cm–1 (DNA), 1397 cm–1 (CH3), 1543 cm–1 (amide II), 1651 cm–1 (amide I), 2924 cm–1 (fatty acids). Additionally, peak shifts were observed at 1122 cm–1 (RNA), 1397 cm–1 (CH3), 1651 cm–1 (amide I), 2851 cm–1 (fatty acids) and 2962 cm–1 (fatty acids). An analysis of the ratio between band areas was conducted, in order to obtain an index that could effectively distinguish between these two cell lines. The following ratios were found: 1650 cm–1/1540 cm–1, 1650 cm–1/1740 cm–1, 1650 cm–1/1084 cm–1 and 1120 cm–1/1084 cm–1. This work demonstrates that it is possible to distinguish between MCF-7 and SKBr3 cells through differences in their FTIR spectra. This work enables distinction between two cell lines from the same breast cancer.