The accumulation of intraneuronal inclusions containing misfolded alpha-synuclein (aSyn) within the central nervous system (CNS) is a common feature found in several neurodegenerative disorders including Parkinson’s disease (PD). Emerging evidence indicates that aSyn amyloid fibrils, a configuration that is present within these characteristic inclusions, are capable of self-replicating by templating the conversion of endogenously expressed aSyn in neurons. Stereotaxic administration of synthetic α-synuclein preformed fibrils (PFFs) into the mouse brain has been shown to seed the formation of intracellular aSyn pathology reminiscent of Lewy body (LB) inclusions present in human PD and related synucleinopathies. Moreover, pathology can be targeted to specific CNS regions. This experimental approach provides a versatile platform for investigating PD-like LB pathology in vivo. We focus here on procedures for initiating aSyn inclusion formation at various regions of the mouse brain using computer-assisted motorized stereotaxic microinjection of aSyn PFFs and discuss appropriate strategies for controls and analysis.