Aims/Purpose: The non‐invasive evaluation of biomarkers for the screening, diagnosis, and monitoring of ocular and neurological diseases is receiving increasing attention. Targeted retinal spectroscopy (TRS) enables simultaneous imaging and high‐quality spectral analysis from specific regions of the eye fundus. It offers valuable information on the structure, composition, and function of retinal tissues. This study demonstrates the capabilities of TRS and assesses its effectiveness in retinal oximetry.Methods: The Zilia Ocular TRS platform was developed and evaluated. First, a reference target and a model eye were used to demonstrate the targeted spectral analysis. Then, Monte Carlo simulations were used to identify crucial TRS parameters—such as spectral range, resolution, noise, and tissue scattering—for retinal oximetry. Simultaneous imaging of the eye fundus and diffuse reflectance spectra acquisitions were performed in two targeted regions of the eye fundus of eight healthy subjects to determine blood oxygen saturation (StO2).Results: Experiments performed with the reference target and model eye showed precise and distinct spectral signatures for each targeted region. Simulation results showed that highest StO2 accuracy was obtained with a spectral range between 530 nm and 585 nm, while a resolution below 4 nm compromised the accuracy. In addition, acquisition areas larger than blood vessels led to an underestimation of StO2, and a linear correlation was found between the additive noise level and the variability of StO2. In vivo oximetry measurements revealed significant differences in StO2 between the optic nerve head and the parafovea.Conclusions: TRS opens up new possibilities for disease screening, diagnosis and monitoring, including assessing oximetry in glaucoma, diabetic retinopathy, age‐related macular degeneration, etc. To account for confounding factors, careful selection of acquisition parameters is crucial, as emphasized in this study.