An alternative approach for optimisation of multiple polymerase chain reaction (PCR) by means of experimental design techniques is described. In initial experiments, the effects of independent variables: deoxyribonucleotides (dNTPs), Mg2+ and primers on multiple PCR were determined by Full Factorial Design. According to the results, each factor (stand-alone or composed ones) influences the amplification of individual genes in multiple PCR in a different way. A three-dimensional Simplex optimisation strategy was used for studying and consequently for improving multiple PCR yields. Using optimised concentrations of dNTPs, Mg2+ and primers in PCR, an improved simultaneous amplification of pkaC and pepC was achieved. Moreover, also in duplex RT-PCR the optimised PCR significantly increased the yield of both transcripts. The outlined optimisation strategy could be adopted to any multiple PCR or RT-PCR.