Advances in precision microirradiators for small animal radiation oncology studies have provided the framework for novel translational radiobiological studies. Such systems target radiation fields at the scale required for small animal investigations, typically through a combination of on-board computed tomography image guidance and fixed, interchangeable collimators. Robust targeting accuracy of these radiation fields remains challenging, particularly at the millimetre scale field sizes achievable by the majority of microirradiators. Consistent and reproducible targeting accuracy is further hindered as collimators are removed and inserted during a typical experimental workflow. This investigation quantified this targeting uncertainty and developed an online method based on a virtual treatment isocenter to actively ensure high performance targeting accuracy for all radiation field sizes. The results indicated that the two-dimensional field placement uncertainty was as high as 1.16 mm at isocenter, with simulations suggesting this error could be reduced to 0.20 mm using the online correction method. End-to-end targeting analysis of a ball bearing target on radiochromic film sections showed an improved targeting accuracy with the three-dimensional vector targeting error across six different collimators reduced from [Formula: see text] mm (mean ± SD) to [Formula: see text] mm for an isotropic imaging voxel size of 0.1 mm.