Abstract Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne New World alphavirus responsible for severe morbidity and mortality in humans and equines. The development of therapeutics against VEEV is critical due to its potential for outbreak and aerosolization as a bioterrorist agent. However, effective and widely accessible antiviral agents for VEEV do not exist. Humoral immunity is an important defense against many alphavirus infections, and we thus generated a panel of 17 highly neutralizing murine VEEV-specific monoclonal antibodies (mAbs) using the VEEV vaccine TC-83 and attenuated chimeric SINV-VEEV Trinidad donkey (TRD) subtype IAB. Nine of these mAbs demonstrate 50% effective inhibitory concentrations (EC50) values below 100 ng/ml when tested against enzootic subtype ID and epizootic VEEV subtypes IAB and IC virion envelope glycoproteins. To define specific viral epitopes important for mAb binding, mapping studies including competition assays, HDX, alanine-scanning mutagenesis, and viral escape mutants are ongoing. Preliminary data suggests the epitopes for these mAbs are found in both the A and B domains of the surface virion envelope 2 glycoprotein. Additionally, several of these mAbs confer prophylactic and therapeutic protection in mice aerosol challenged with virulent TRD VEEV. Mechanism of action studies are underway to define the steps in the virus life cycle (viral attachment, entry, fusion, and egress) at which our most potent protective antibodies act. Identification of specific epitopes targeted by these protective mAbs will better inform the development of targeted immunotherapeutics and vaccines against VEEV and related encephalitic alphaviruses.