Live cell Raman micro-spectroscopy is emerging as a promising bioanalytical technique for label-free discrimination of a range of different cell types (e.g. cancer cells and fibroblasts) and behaviors (e.g. apoptosis). The aim of this study was to determine whether confocal Raman micro-spectroscopy shows sufficient sensitivity and specificity for identification of primary human bronchial epithelial cells (HBECs) to be used for live cell biological studies in vitro. We first compared cell preparation substrates and media, considering their influence on lung cell proliferation and Raman spectra, as well as methods for data acquisition, using different wavelengths (488 nm, 785 nm) and scan protocols (line, area). Evaluating these parameters using human lung cancer (A549) and fibroblast (MRC5) cell lines confirmed that line-scan data acquisition at 785 nm using complete cell media on a quartz substrate gave optimal performance. We then applied our protocol to acquisition of data from primary human bronchial epithelial cells (HBEC) derived from three independent sources, revealing an average sensitivity for different cell types of 96.3% and specificity of 95.2%. These results suggest that Raman micro-spectroscopy is suitable for delineating primary HBEC cell cultures, which in future could be used for identifying different lung cell types within co-cultures and studying the process of early carcinogenesis in lung cell culture.