Organotypic cultures from the ventral mesencephalon (VM) are widely used to model Parkinson’s disease (PD). In this method, neurotoxic compounds have traditionally been applied to the media to induce a uniform dopaminergic (DAergic) cell death in the tissue slices, regardless of the variation existing among slices. This study demonstrates a refinement of the toxic induction technique. We show that unilateral application of 6-hydroxydopamine (6-OHDA) at the tissue surface by means of a microelectrode causes a precisely localized cell death that closely resembles an in vivo stereotactic model. This technique introduces an internal control that accounts for variation between slices and enables a precise quantification of the cell loss due to the toxin in use. We characterized organotypic VM cultures in terms of effects of 6-OHDA toxicity and number of DAergic neurons as judged by immunofluorescence and Western blots. Our findings illustrate that this new application technique greatly improves the representativeness of organotypic cultures as a model for PD.