Pathogenic mutations in Leucine-rich repeat kinase 2 (LRRK2), such as G2019S, represent the predominant genetic cause of Parkinson's disease (PD), frequently resulting in heightened kinase activity. Consequently, LRRK2 kinase inhibitors have emerged as promising treatments for PD. Despite ongoing clinical and pre-clinical trials of these inhibitors, specific, sensitive, and non-invasive biomarkers for measuring drug target engagement or stratifying mutation carriers remain elusive. Utilizing our scalable and sensitive Orbitrap Exploris 480 mass spectrometer connected to an EASY-nLC 1200 system, we reproducibly quantified over 3,000 proteins in urine samples obtained from LRRK2 knock-out and G2019S rats, as well as from over 1,000 individuals with and without LRRK2 mutations across multiple independent cohorts. Loss of LRRK2 and pathogenic mutations led to significant changes in the levels of 94 proteins associated with immune processes and lysosome-glycosphingolipid metabolism in rat urine. To classify individuals as bearing LRRK2 mutations or not, we employed a random forest model that narrowed down the list of 94 significantly regulated rat proteins to the 22 most important features in humans, thereby defining a panel of LRRK2 activity markers. Interestingly, the machine learning model trained using these features excelled in classifying the mutation status in the largest human urine cohort (Parkinson"s Progression Markers Initiative - PPMI[MOU1] : 634 WT and 343 G2019S), achieving an AUC of 0.86 and an average precision of 0.91. Some of the panel proteins, such as ENPEP, FUCA1, and CTSZ, also correlated strongly with disease severity scores such as UPDRS and the Hoehn and Yahr PD progression score, underscoring the multimodality of these proteins in evaluating the severity and stratification of familial PD. Lastly, we further validated the utility of our biomarker panel by demonstrating that the elevated levels of marker proteins in the urine of rats with the LRRK2 G2019S mutation were ameliorated in the urine of rats treated with LRRK2 inhibitors. Together, our study underscores the potential of proteomics for identifying LRRK2 biomarkers and for the non-invasive evaluation of LRRK2 inhibitor efficacy in human urine, thereby aiding in the development of therapeutics for PD.