Design of experiments (DoE) concept was successfully applied to determine the optimum processing conditions that yield maximum % exhaustion for berberine interaction with synthetic tanning agent pretreated polyamide substrates. The potential of synthetic tanning agent to provide anionic sites on the polyamide for berberine interaction which is cationic in nature was tested to increase the % exhaustion of berberine in this article. Experiments were designed according to Central Composite Rotatable Design (CCRD). The three factors for synthetic tanning agent pretreatment and two factors for berberine interaction each at five different levels, including central and axial points were considered. Experiments were conducted in a laboratory scale infra-red treatment instrument according to CCRD. For each response, second order polynomial models were developed using multiple linear regression analysis incorporating linear, interactions and squared effects of all variables and then optimized. The significance of the mathematical model developed was ascertained using Excel regression (solver) analysis module. Analysis of variance (ANOVA) was performed to check the adequacy and accuracy of the fitted models. The response surfaces and contour maps showing the interaction of process variables were constructed. Applying Monte Carlo simulation, response surface and contour plots, optimum operating conditions were found and at this optimum point, % exhaustion of 81% and 74% respectively for synthetic tanning agent pretreatment and berberine interaction were observed and subsequently the results were experimentally investigated.