In the present study, drug-in-adhesive matrix type combinational prophylactic transdermal patch composed of eserine and pralidoxime chloride against acetyl choline agonist (±)-anatoxin a neurotoxin poisoning was developed. initially, a simple rp-hplc method was developed and validated for the simultaneous determination and quantification of eserine and 2-pam using uv detection. the method was validated as per ich guidelines for validation of analytical procedures, and was applied for the routine analysis of these two drugs in fabricated transdermal patches. adhesive matrix type transdermal patches containing eserine and 2-pam were prepared by solvent casting method. the drug combinations were incorporated in adhesive matrix type system supported by a polyester film laminate backing membrane and attached to a temporary release-liner. the dermal patches was having desired properties such as thin, circular, opaque, smooth, homogeneous, sticky, uniform, flat and flexible in nature. the drug release was sustained from all the formulations up to 72 h and following anomalous (non-fickian) diffusion and fickian release mechanism for eserine and 2-pam, respectively. the release kinetic for both drugs follows higuchi model kinetics. optimized transdermal patch exhibited highest acceptable levels of tackiness with good adherence capacity and showed promising stability potential with respect to all points of analysis. from safety point of view, the optimized transdermal patch was safe for application to the skin with no dermal and mutagenic toxicity as well. pharmacodynamic study proved that the optimized transdermal patch was effective against acetyl choline agonist (±)-anatoxin a neurotoxin poisoning. pharmacokinetic study revealed that the systemic absorption of the drugs from the fabricated best optimized patch through the skin was sufficient enough to achieve pharmacodynamic efficacy. finally, radio-scintigraphy study assured the localization of radiolabel eserine and 2-pam from adhesive matrix type transdermal patch to the targeted organ (brain). in these regards, this fabricated drug-in-adhesive matrix type combinational prophylactic transdermal patch composed of eserine and pralidoxime chloride is of particular clinical significance for providing prophylactic efficacies against acetyl choline agonist (±)-anatoxin a neurotoxin poisoning in biological warfare situations.