Increased leucine-rich repeat kinase 2 (LRRK2) kinase activity is an established risk factor for Parkinson's disease (PD), and several LRRK2 kinase inhibitors are in clinical development as potential novel disease-modifying therapeutics. This biomarker characterization study explored within- and between-subject variability of multiple LRRK2 pathway biomarkers (total LRRK2 [tLRRK2], phosphorylation of the serine 935 (Ser935) residue on LRRK2 [pS935], phosphorylation of Rab10 [pRab10], and total Rab10 [tRab10]) in different biological sources (whole blood, peripheral blood mononuclear cells [PBMCs], neutrophils) as candidate human target engagement and pharmacodynamic biomarkers for implementation in phase I/II pharmacological studies of LRRK2 inhibitors. PD patients with a LRRK2 mutation (n = 6), idiopathic PD patients (n = 6), and healthy matched control subjects (n = 10) were recruited for repeated blood and cerebrospinal fluid (CSF) sampling split over 2 days. Within-subject variability (geometric coefficient of variation [CV], %) of these biomarkers was lowest in whole blood and neutrophils (range: 12.64%-51.32%) and considerably higher in PBMCs (range: 34.81%-273.88%). Between-subject variability displayed a similar pattern, with relatively lower variability in neutrophils (range: 61.30%-66.26%) and whole blood (range: 44.94%-123.11%), and considerably higher variability in PBMCs (range: 189.60%-415.19%). Group-level differences were observed with elevated mean pRab10 levels in neutrophils and a reduced mean pS935/tLRRK2 ratio in PBMCs in PD LRRK2-mutation carriers compared to healthy controls. These findings suggest that the evaluated biomarkers and assays could be used to verify pharmacological mechanisms of action and help explore the dose-response of LRRK2 inhibitors in early-phase clinical studies. In addition, comparable α-synuclein aggregation in CSF was observed in LRRK2-mutation carriers compared to idiopathic PD patients.