Stereotactic neurosurgery is used in pre-clinical research of neurological and psychiatric disorders in experimental rat and mouse models to engraft a needle or electrode at a pre-defined location in the brain. However, inaccurate targeting may confound the results of such experiments. In contrast to the clinical practice, inaccurate targeting in rodents remains usually unnoticed until assessed by ex vivo end-point histology. We here propose a workflow for in vivo assessment of stereotactic targeting accuracy in small animal studies based on multi-modal post-operative imaging. The surgical trajectory in each individual animal is reconstructed in 3D from the physical implant imaged in post-operative CT and/or its trace as visible in post-operative MRI. By co-registering post-operative images of individual animals to a common stereotaxic template, targeting accuracy is quantified. Two commonly used neuromodulation regions were used as targets. Target localization errors showed not only variability, but also inaccuracy in targeting. Only about 30% of electrodes were within the subnucleus structure that was targeted and a-specific adverse effects were also noted. Shifting from invasive/subjective 2D histology towards objective in vivo 3D imaging-based assessment of targeting accuracy may benefit a more effective use of the experimental data by excluding off-target cases early in the study.Stereotactic neurosurgery is used to introduce a needle or an electrode at a precise location in the brain, for instance to perform deep brain stimulation (DBS), lesioning (e.g. ablation) or cell-based therapy in patients to ameliorate symptoms of neurological disease. Clinical use of stereotactic neurosurgery benefits from experimental research on rodent models, for instance to identify potential target regions. Likewise, small animal investigations like DBS for neuromodulation 1,2 , electrochemical/ electrolytic lesioning for creating animal models or ameliorating symptoms 3,4 , as well as gene therapy investigations involving injection of stem cells/therapeutic agents/tracers 5-7 requires stereotactic brain surgery. They rely on standard stereotaxic brain atlases (e.g. The rat brain in stereotaxic coordinates (George Paxinos and Charles Watson, Academic Press, 2006 8 ) and skull landmarks (e.g. bregma (B), lambda (L)) for localizing the target in the rodent brain and for surgery planning 9 . Stereotaxic frames used in small animal brain surgery have species-specific head mounting platforms, with a manipulator that can move in three dimensions along the axes with respect to the head holder. The targeting accuracy of such devices is dependent on numerous factors, such as inter-animal anatomical variability, positioning errors (e.g. skull flat position), scaling errors (e.g. when using the same atlas with animals of a different size or strain), errors in intra-operative localization of bregma, operator-specific deviations etc. Some of these sources of inaccuracy in small animal stereotactic surgery have been characterized in previo...