Our dual wavelength atlas approach to spectral unmixing has the potential to differentiate two chromophores using only two optical wavelengths. This work investigates the optimization of our method by varying six parameters. An iterative process was used to determine the optimal set of parameters by changing each parameter one at a time and finding which value returned the highest combination of sensitivity and specificity. Once the optimal set of parameters were applied, our novel dual-wavelength atlas method resulted in 10% higher balanced accuracy. These results are promising for photoacoustic-guided surgery.