In this paper a method for isolation and purification of the pertussis toxin from the cell culture of Bordetella pertussis was presented. Pertussis toxin production was enhanced with the optimization of the microbiological media and cultivation time. Relatively high degree of purification of 80,39% and yield of 56% was resulted by the unique combination of the protein isolation and purification methods. Using immunoelectrophoresis and high performance liquid chromatography, in comparison with pertussis toxin WHO standard, it was verified that the protein isolated is indeed pertussis toxin. The conclusion of this paper is that this unique combination of the different techniques along with the specific modifications implemented would give good results in pertussis toxin isolation and purification on laboratory level, while scaling up of this methodology could be the objective of future research. It can also be concluded that, no matter what literature data say and what opinions other authors have, all methods and working conditions should be examined and optimized for the reason of the specificity of the autochthonous species as well as the dynamic changes in this field.