Introduction: Severe motoric symptoms in parkinson’s disease arise from the loss of dopaminergic input into the striatum. The striatal efferent nuclei are thus affected by a lack of GABAergic input. Deep brain stimulation (DBS) in the subthalamic nucleus (STN) seems to counterbalance this effect by a hitherto unknown molecular mechanism. Based on the hypothesis that high frequency electrical stimulation may have an effect on neurotransmitter release in the local environment of the stimulation electrode we established an in vivo rat model allowing for simultaneous and collocated high frequency stimulation and microdialysis in freely moving rats. Methods: Using standard stereotaxic techniques we implanted a double tube guiding cannula right above the corresponding target nucleus and fixed it on the skull for permanent disposition. After a 7-day period of recovery we inserted a microdialysis probe and a stimulation electrode into the guiding cannula such that the tips of the probes were placed in the center of the target nucleus. The electrode surface was pointing to the microdialysis membrane with a distance smaller than 1 mm. Thus we were able to sample neurotransmitter outflow during the periods of electrical high frequency stimulation in different target nuclei. After the experiment we removed the brain and examined the accuracy of the probe placement using histological slices of the rat brain. Problem: Though leading to promising results in the rat caudate nucleus with a targeting accuracy of 100% we had to face problems with the placement of stimulation electrode and microdialysis membrane into the small sized rat STN using standard stereotaxic techniques. Solution: We employed a new robot-assisted neuronavigation framework for small animal stereotaxy. The robotized spherical assistant for stereotaxic surgery (SASSU) allows automated, precise and repeatible implantation of probes into the brain. It is controlled by a navigation software based on coronal slices of the rat brain. Therefore, software assisted preoperative planning could be easily performed using the software. Bregma and Lambda were used as landmarks for the registration of the rat skull like it is done with the standard stereotaxic methods. Software parameter definition took place according to the surgical workflow. The SASSU step size and velocity were adjusted on demand. By definition of five different parameters we were able to guide a microdialysis probe and a stimulation electrode near together into the rat STN with any specified angle of entry and a positioning accuracy smaller than 45 μm. The SASSU therefore provides a new stereotaxic tool for target optimization in DBS of the rat STN.