Background and Objectives: Accurate identification of nerves is critical to ensure safe and effective delivery of regional anesthesia during peripheral nerve blocks. Nerve stimulation is commonly used, but it is not perfect. Even when nerve stimulation is performed in conjunction with ultrasound guidance, determining when the needle tip is at the nerve target region can be challenging. In this in vivo pilot study, we investigated whether close proximity to the brachial plexus and penetration of the axillary artery can be identified with optical reflectance spectroscopy, using a custom needle stylet with integrated optical fibers. Methods: Ultrasound-guided insertions to place the needle tip near the brachial plexus at the axillary level were performed at multiple locations in 2 swine, with the stylet positioned in the cannula of a 20-gauge stimulation needle. During each insertion, optical reflectance spectra were acquired with the needle tip in skeletal muscle, at the surface of muscle fascia, and at the nerve target region; confirmation of the final needle position was provided by nerve stimulation. In addition, an insertion to the lumen of the axillary artery was performed in a third swine. Differences in the spectra were quantified with lipid and hemoglobin parameters that provide contrast for optical absorption by the respective chromophores. Results: The transition of the needle tip from skeletal muscle to the nerve target region was associated with higher lipid parameter values (P <0.001) and lower hemoglobin parameter values (P <0.001). The transition of the needle tip from muscle fascia to the nerve target region was associated with higher lipid parameter values (P = 0.001). Intraluminal access of the axillary artery was associated with an elevated hemoglobin parameter. Conclusions: Spectroscopic information obtained with the optical needle is distinct from nerve stimulation and complementary to ultrasound imaging, and it could potentially allow for reliable identification of the injection site during peripheral nerve blocks.