Simple, precise and accurate zero order derivative spectroscopic method has been developed and validated for the estimation of dalfampridine in bulk and pharmaceutical dosage form. The drug shows maximum absorption (λ max) at 262nm in 0.1M HCL and obeys Beer’s law in the concentration range of 0.5-3.0µg/ml. The linearity study was carried out and regression coefficient was found to be 0.9976 and it has showed good linearity, precision during this concentration range. The % recovery was found to be 98.66% -101.56%. The LOD and LOQ were found to be 0.0360 and 0.109µg/ml. The % relative standard deviation were found to be less than 2. According to ICH guidelines the technique has been validated for linearity, precision, accuracy, ruggedness, LOD and LOQ. The developed and validated method can be successfully applied for routine quantification of Dalfampridine in bulk and pharmaceutical dosage form.