Parkinson’s disease (PD) is a common neurodegenerative disease with multi-factorial etiopathogenesis involves in the degeneration of dopaminergic neurons. Thus, the discovery of drug candidates acting on new targets of PD are required to address the varied pathological aspects and modify the disease process. This study was to target Rho-associated protein kinase (ROCK) to identify new neuroprotectants to tackle brain disorders including PD using combined computational and phenotypic screening-based drug discovery approaches. Two small molecule compounds, 2-(5-methyl-1-benzofuran-3-yl)-N-(5-propylsulfanyl-1, 3, 4-thiadiazol-2-yl)acetamide (MBPTA) and Benzyl 7-(4-hydroxy-3- methoxyphenyl)-5-methyl-4,7- dihydrotetrazolo[1,5-a]pyrimidine-6-carboxylate (BHDPC) have been identified for inhibition of ROCK activity with significant protective effects against 1-methyl-4-phenylpyridinium ion (MPP+ )-induced damage in multiple in vitro and in vivo models. The present study is to investigate their pharmacologic effects and the underlying mechanisms of actions in multiple experimental MPP+ /MPTP -induced PD models. Pre-treatment of neuroblastoma SH-SY5Y cells with MBPTA significantly suppressed MPP+ -induced cells death by restoring abnormal changes in numerous apoptotic regulators. MBPTA was able to inhibit MPP+ -induced ROS/NO generation, NF-κB/iNOS pathway, indicating the critical role of MBPTA in regulating ROS/NOmediated cell death. Furthermore, MBPTA was shown to activate PI3K/Akt survival signaling and its cytoprotective effect was abolished by PI3K and Akt inhibitors. These results suggest that MBPTA protects against MPP+ -induced apoptosis in the neuronal cell line through inhibition of ROS/NO generation and activation of PI3K/Akt signaling. BHDPC exhibitedthe protective effects against MPP+ neurotoxicity in human neuroblastoma SH-SY5Y cells. BHDPC was able to up-regulate PKA phosphorylation and increased CREB-mediated Bcl2 expression and its cytoprotective effect was abolished by PKA inhibitor. Further study in rat organotypic cerebellar cultures indicated that BHDPC was able to suppress MPP+ - induced neuronal cells death. In MPTP-induced Parkinsonism model of zebrafish, BHDPC attenuated dramatically the MPTP-induced dopaminergic neurons loss and behavior movement deficiency, supporting its potential neuroprotective activity in in vivo. Taken together, our results revealed that BHDPC provided the pro-survival effects in multiple models of PD in vitro and in vivo. Collectively, MBPTA and BHDPC, represent promising lead candidates for the treatment of PD, through different mechanisms.