Endoscopic surveillance is recommended for patients with Barrett's esophagus (BE) to detect high-grade intraepithelial neoplasia (HGIN) or early cancer (EC). Early neoplasia is difficult to detect with white light endoscopy and random biopsies are associated with sampling error. Fluorescence spectroscopy has been studied to distinguish non-dysplastic Barrett's epithelium (NDBE) from early neoplasia. The Optical Biopsy System (OBS) uses an optical fiber integrated in a regular biopsy forceps. This allows real-time spectroscopy and ensures spot-on correlation between the spectral signature and corresponding physical biopsy. The OBS may provide an easy-to-use endoscopic tool during BE surveillance. We aimed to develop a tissue-differentiating algorithm and correlate the discriminating properties of the OBS with the constructed algorithm to the endoscopist's assessment of the Barrett's esophagus. In BE patients undergoing endoscopy, areas suspicious for neoplasia and endoscopically non-suspicious areas were investigated with the OBS, followed by a correlating physical biopsy with the optical biopsy forceps. Spectra were correlated to histology and an algorithm was constructed to discriminate between HGIN/EC and NDBE using smoothed linear dicriminant analysis. The constructed classifier was internally cross-validated and correlated to the endoscopist's assessment of the BE segment. A total of 47 patients were included (39 males, age 66 years): 35 BE patients were referred with early neoplasia and 12 patients with NDBE. A total of 245 areas were investigated with following histology: 43 HGIN/EC, 66 low-grade intraepithelial neoplasia, 108 NDBE, 28 gastric or squamous mucosa. Areas with low-grade intraepithelial neoplasia and gastric/squamous mucosa were excluded. The area under the receiver operating characteristic curve of the constructed classifier was 0.78. Sensitivity and specificity for the discrimination between NDBE and HGIN/EC of OBS alone were 81% and 58% respectively. When OBS was combined with the endoscopist's assesssment, sensitivity was 91% and specificity 50%. If this protocol would have guided the decision to obtain biopsies, half of the biopsies would have been avoided, yet 4/43 areas containing HGIN/EC (9%) would have been inadvertently classified as unsuspicious. In this study, the OBS was used to construct an algorithm to discriminate neoplastic from non-neoplastic BE. Moreover, the feasibility of OBS with the constructed algorithm as an adjunctive tool to the endoscopist's assessment during endoscopic BE surveillance was demonstrated. These results should be validated in future studies. In addition, other probe-based spectroscopy techniques may be integrated in this optical biopsy forceps system.