The physico-chemical environment of the separation, applied on a complex peptide hydrolysate obtained from the hydrolysis of bovine hemoglobin by pepsin, to recover an antimicrobial peptide fraction was shown to influence its recovery efficiency and selectivity. The objective of the present work was to evaluate experimental conditions that maximize the adsorption capacity of the antimicrobial peptide fraction on liquid/air interface through the use of an experimental design methodology. A first step comprised a study on several factors thanks to a screening matrix to acquire basic knowledges on these variables and their influence on the extraction selectivity. Hydrolysate and salt concentrations, pH of both the hydrolysate and draining solutions were established as the four main factors. Then a composite matrix was studied to identify with response surface modelling the optimum conditions for fractionation.