Objectives: We recently introduced a navigated, robot-driven laser beam craniotomy for use with stereoelectroencephalography (SEEG) applications. This method was intended to substitute the hand-held electric power drill in an ex vivo study. The purpose of this in vivo non-recovery pilot study was to acquire data for the depth control unit of this laser device, to test the feasibility of cutting bone channels, and to assess dura perforation and possible cortex damage related to cold ablation.Methods: Multiple holes suitable for SEEG bone channels were planned for the superior portion of two pig craniums using surgical planning software and a frameless, navigated technique. The trajectories were planned to avoid cortical blood vessels using magnetic resonance angiography. Each trajectory was converted into a series of circular paths to cut bone channels. The cutting strategy for each hole involved two modes: a remaining bone thickness mode and a cut through mode (CTR). The remaining bone thickness mode is an automatic coarse approach where the cutting depth is measured in real time using optical coherence tomography (OCT). In this mode, a pre-set measurement, in mm, of the remaining bone is left over by automatically comparing the bone thickness from computed tomography with the OCT depth. In the CTR mode, the cut through at lower cutting energies is managed by observing the cutting site with real-time video.Results: Both anesthesia protocols did not show any irregularities. In total, 19 bone channels were cut in both specimens. All channels were executed according to the planned cutting strategy using the frameless navigation of the robot-driven laser device. The dura showed minor damage after one laser beam and severe damage after two and three laser beams. The cortex was not damaged. As soon as the cut through was obtained, we observed that moderate cerebrospinal fluid leakage impeded the cutting efficiency and interfered with the visualization for depth control. The coaxial camera showed a live video feed in which cut through of the bone could be identified in 84%.Conclusion: Inflowing cerebrospinal fluid disturbed OCT signals, and, therefore, the current CTR method could not be reliably applied. Video imaging is a candidate for observing a successful cut through. OCT and video imaging may be used for depth control to implement an updated SEEG bone channel cutting strategy in the future.