Introduction: A significant number of transthoracic diagnostic biopsy procedures for lung lesions show indeterminate results. Such failures are potentially due to inadequate recognition of vital tumor tissue. The objective of this study was to evaluate whether optical spectroscopy at the tip of a biopsy needle device can improve the accuracy of transthoracic lung biopsies. Methods: Ex vivo optical measurements were performed on lung tissue from 13 patients who underwent either lobectomy or segmental resection for primary non-small cell lung cancer or pulmonary metastases from various origins. From Diffuse Reflectance Spectroscopy (DRS) and Fluorescence Spectroscopy (FS) measurements, different parameters were derived such as tissue composition as well as physiological and metabolic characteristics. Subsequently, a classification and regression trees (CART) algorithm was used to classify the type of tissue based on the derived parameters. Histology analysis was used as gold standard to report sensitivity and specificity of the tissue classification based on the present optical method. Results: Collective analysis of all DRS measurements showed an overall discrimination between lung parenchyma and tumor tissue with a sensitivity and specificity of 98 and 86%, respectively. When the data were analyzed per individual patient, eliminating inter-patient variation, 100% sensitivity and specificity was achieved. Furthermore, based on FS parameters, necrotic and non-necrotic tumor tissue could be distinguished with 91% sensitivity and specificity. Conclusion: This study demonstrates that DRS provides accurate diagnosis of malignant lung lesions, whereas FS enables identification of necrotic tissue. When both optical techniques are combined within a biopsy device, the diagnostic performance and the quality of transthoracic biopsies could significantly be enhanced.