Brain tumor needle biopsies are performed to retrieve tissue samples for neuropathological analysis. Although preoperative images guide the procedure, there are risks of hemorrhage and sampling of non-tumor tissue. This study aimed to develop and evaluate a method for frameless one-insertion needle biopsies with in situ optical guidance and present a processing pipeline for combined postoperative analysis of optical, MRI, and neuropathological data. An optical system for quantified feedback on tissue microcirculation, gray–whiteness, and the presence of a tumor (protoporphyrin IX (PpIX) accumulation) with a one-insertion optical probe was integrated into a needle biopsy kit that was used for frameless neuronavigation. In Python, a pipeline for signal processing, image registration, and coordinate transformation was set up. The Euclidian distances between the pre- and postoperative coordinates were calculated. The proposed workflow was evaluated on static references, a phantom, and three patients with suspected high-grade gliomas. In total, six biopsy samples that overlapped with the region of the highest PpIX peak without increased microcirculation were taken. The samples were confirmed as being tumorous and postoperative imaging was used to define the biopsy locations. A 2.5 ± 1.2 mm difference between the pre- and postoperative coordinates was found. Optical guidance in frameless brain tumor biopsies could offer benefits such as quantified in situ indication of high-grade tumor tissue and indications of increased blood flow along the needle trajectory before the tissue is removed. Additionally, postoperative visualization enables the combined analysis of MRI, optical, and neuropathological data.