Parkinson's disease (PD) is the most common neurodegenerative motor disorder and primarily affects movement control but also a range of non-motor functions. With unknown etiology and lack of cure, much research is dedicated to unravel pathological mechanisms and improve clinical prospects for symptom alleviation, prevention and treatment. To achieve these goals, animal models intended to represent symptoms similar to those observed in the complex clinical display of PD play a key role. It is important to bear in mind that, in any studies with laboratory animals, it is crucial to take the 3Rs principle (Refine, Reduce, Replace) into account. The main pathology of PD includes degeneration of dopamine neurons in the substantia nigra pars compacta (SNc). The 6-hydroxydopamine (6-OHDA) lesion model, in which dopaminergic neurons are chemically destroyed, is often favored as a laboratory model of PD in both rodents and primates. However, while reproducing several features of clinical PD, mice exposed to 6-OHDA frequently experience systemic dysfunction causing premature death. To avoid suffering and unnecessary deaths of laboratory mice, there is a need for improved experimental protocols in accordance with the 3Rs principle. Based on current literature and our own previous experiments, we decided to test the effect of three parameters: 1) reduced dose of the 6-OHDA toxin; 2) daily post-operative care to avoid hypothermia and energy loss; 3) shortened interval from surgical injection of toxin to time of sacrifice. By implementing a 6-OHDA lesion protocol using a lower dose of toxin than commonly seen in the literature alongside careful post-operative care and decreased time post-injection, a fully recovered weight post-surgery and high survival rate was obtained. This was achieved despite full expression of the 6-OHDA-induced locomotor phenotype. A step-by-step protocol was formulated. Validation using histological analysis confirmed toxin-induced degeneration of midbrain dopamine neurons with concomitant loss of dopaminergic projections in the lesioned hemisphere. Notably, while SNc dopamine neurons were drastically reduced, those located in the ventral tegmental area (VTA) were less affected in a medialhigh survival to laterallow survival manner. The Refine and Reduce parameters of the 3Rs principle in experimental animal welfare were specifically addressed which allowed us to improve well-being and survival of mice while maintaining characteristic parkinsonian features in the 6-OHDA lesion model. A table summarizing current literature on the 6-OHDA model in rodents and our validated step-by-step experimental protocol are provided.