A colorimetric nano-platform is reported for phenobarbital determination in human plasma samples. It is based on the inhibitory effect of phenobarbital on melamine induced aggregation of sodium dodecyl sulfate modified silver nanoparticles. The interaction of melamine and phenobarbital results in the disaggregation of nanoparticles and consequently increase the absorbance intensity proportional to phenobarbital concentration, and color change from purple to yellow. A central composite design as a multivariate optimization approach with four independent parameters (i.e. pH, melamine concentration, nanoparticle concentration and time) was employed to optimize the reaction conditions. In the optimized conditions, the presented method was fully validated by investigation of system linearity, sensitivity, accuracy, precision, and selectivity. The validated method was used for the determination of phenobarbital in plasma samples collected from patients receiving phenobarbital.