Introduction and Aim: Parkinson's disease (PD) is a neurodegenerative disorder that destroys dopaminergic neurons in the nigrostriatal area of the midbrain this results in a movement difficulty characterised by tremors, bradykinesia, and postural instability The present in vitro study is designed to assess the beneficial (cytoprotectant and anti-inflammatory) and cyto-toxicological effects of ethanolic Withania somnifera (Ashwagandha) root extract (EWSR) in presence of Rotenone (ROT) and other known neuroprotectants (Doxycycline and Ellagic acid).   Materials and Methods: The nine groups used in the in vitro studies were normal control, positive control (Rotenone or Rot), W. somnifera root extract (EWSR), Doxycycline (Doxy), Ellagic acid (EA), ROT with EWSR, ROT with doxy, ROT with EA, ROT with EWSR-doxy, and ROT with EWSR-doxy-EA. The EWSR, doxy and EA treatment was started on the 24th hour following the rotenone administration and continued for the next 36 hours. After the 36th hour of protective therapy, quantification of pro-inflammatory cytokines (ELISA), cell viability or analyses of neuronal cell loss, cell cycle analysis and molecular analysis (RTPCR) was performed as a part of assessment.   Results: Observations from the present in vitro studies clearly demonstrate the cytoprotective and anti-inflammatory properties of EWSR, and these beneficial or protective effects got enhanced when treated in combination with Doxy and EA indicating synergistic effect in the cells that were priorly treated with ROT, and later treated with EWSR, Doxy, and EA individually and in combination.   Conclusion: The findings imply that EWSR reduces the in vitro effects of ROT by acting as an anti-inflammatory and cytoprotective agent.