Coccidiosis, a disease caused by parasites of the genus Eimeria, is the major pathogenic disease in the poultry industry with associated costs of over $3 billion USD annually. Live vaccination can control coccidiosis but its successful implementation can be difficult. Vaccination uses live, infective parasites to establish self-limiting and subclinical infection that stimulates development of robust protective immunity. Accurate dosage is paramount to vaccine success; administration of too many infective parasites will negatively impact bird health while administration of too few will fail to stimulate protective immunity. Vaccine potency is not static and is subject to many variables. Each vaccine lot must be tested in live animals so infectivity and correct dosage can be confirmed. Infection trials are time consuming, expensive, and only semi-quantitative, at best. We have demonstrated a prototype assay that provides rapid assessment of parasite viability (i.e. vaccine potency) based on measurement of actual parasite molecular activity. We have developed a protocol to measure biomolecules produced by vaccine parasite constituents upon exposure to specific stimuli, and shown that the abundance of these specific assay targets reflects actual parasite viability. Ongoing work aims to improve assay accuracy by identifying optimized biomolecule targets and to streamline the assay for ease of use. Additional work supporting assay development will further improve the accessibility and feasibility of vaccination by developing standardized molecular tools for rapid parasite species and strain identification (useful in identifying points of vaccine failure), and by extending the shelf-life of stored vaccines. Our optimized viability assay will measure the precise viability of the constituents of live coccidiosis vaccines in several hours (as opposed to the 10+ days required for standard infection trials). This, combined with the above-mentioned molecular tools we are developing in support of the assay, will greatly improve the efficacy and accessibility of coccidiosis vaccines. This will maximize production efficiencies, profit for farmers, and sustainability of this globally important food-production industry, while minimizing its environmental impact. Improving the efficacy of coccidiosis vaccines will reduce the application of antimicrobial drugs in farming: an important step towards preventing the development of antimicrobial resistance, protecting biodiversity and responding to the demand of Ontario consumers for antibiotic-free agricultural products.