The techniques of fluorescence-guided biopsies were developed in order to overcome the possible limitations of intraoperative histopathological examination. Fluorescence-guided biopsies rely on the administration of a fluorophore, mainly 5-aminolevulinic acid (5-ALA) or fluorescein sodium, before tumor sampling. Then, tumor samples are illuminated under an operative microscope or an endoscope. Fluorescence, if present, warrants that the quality of the samples is good enough to obtain a diagnosis. 5-ALA and fluorescein sodium have a high sensibility and a good predictive positive value but lack specificity. However, the diagnostic performances of the technique vary depending on the histological nature of the tumor, reliability being stronger for high-grade gliomas and lymphomas. No significant side effect is attributed to the technique. Thus, fluorescence-guided biopsy is a safe and time-effective technique that represents a possible alternative to intraoperative histopathological examination, notably in centers where this latter technique is not available.