Clostridium perfringens toxins play role in causing pulpy kidney disease poisoning as well enterotoxaemia. To combat antimicrobial resistance: curbing use, regulations and execution to antimicrobial usage in food along with withdrawal period is necessary. Aim of study was to optimize the toxins production by indigenously characterized C. perfringens type D isolates (n=03) under various physicochemical parameters, a lead towards local vaccine production in Pakistan. Indigenous isolates were characterized on the basis of 16S rRNA and MW349974.1, MW341428.1, MW332258.1 accession numbers were received from NCBI GenBank. Isolates were identified as toxinotype D through PCR toxinotyping. Quinolones antibiotic susceptibility testing revealed that isolates observed susceptible to enrofloxacin and resistant to ciprofloxacillin and ofloxacillin. Optimization of toxins production was determined under the influence of physical and chemical parameters. Alpha and epsilon toxin production in reinforced clostridial medium (RCM) broth was observed higher at 37°C after 24h incubation by MW332258.1. Under the influence of 0.2% glucose and 0.3% tween 80 supplementation in RCM, greater production of alpha and epsilon toxin units was observed by MW332258.1. Under optimized physicochemical parameters, maximum toxins units were observed; MW332258.1 isolate is excellent candidate could be used to produce maximum toxin units for vaccine production at industrial scale.