Abstract: Methods for increasing multiplex level by externalization of a passive reference in polymerase chain reactions (PCR) are provided. An exemplary method comprises providing a first mastermix including a passive fluorescence dye in at least a first well of a plate; providing a second mastermix including an active fluorescence dye in at least a second well of the plate; wherein the passive fluorescence dye and the active fluorescence dye emit a same spectrum and an intensity of the spectrum is adapted to be measured; and wherein the first mastermix is devoid of an active fluorescence dye emitting the same spectrum and the second mastermix is devoid of the passive fluorescence dye emitting the same spectrum. Numerous other aspects are provided.