A method for detection of a plurality of target nucleic acids in a single sample container comprising the steps of: (a) contacting a sample suspected of containing a plurality of target nucleic acids with (i) a first primer/probe set including a first labeled probe and a first primer, and (ii) a second primer/probe set including a second labeled probe and a second primer, wherein said first primer anneals to a target nucleic acid of said plurality of target nucleic acids at a first temperature and said second primer anneals to an additional nucleic acid of said plurality of said target nucleic acids at a second temperature and said first temperature is higher than said second temperature; (b) amplifying said plurality of target nucleic acids in the sample in an amplification reaction that includes at least a two-step annealing/elongation temperature profile including a first step comprising said first temperature and a second step comprising said second temperature; (c) detecting, sequentially, over at least a portion of said annealing/elongation temperature profile (i) a first dectable signal during said first step, which results from annealing/elongation of the first primer and which corresponds to the signal emitted by said first label; (ii) a second detectable signal during said second step, which results from annealing/elongation of the first and second primers and which corresponds to the signal emitted by said first and said second label; and (iii) subtracting said first detectable signal from said second detectable signal to identify the detectable signal emitted by said second label; and (d) measuring a relative amount of each target nucleic acid in said plurality of target nucleic acids. (a) contacting a sample suspected of containing a plurality of target nucleic acids with (i) a first primer/probe set including a first labeled probe and a first primer, and (ii) a second primer/probe set including a second labeled probe and a second primer, wherein said first primer anneals to a target nucleic acid of said plurality of target nucleic acids at a first temperature and said second primer anneals to an additional nucleic acid of said plurality of said target nucleic acids at a second temperature and said first temperature is higher than said second temperature; (b) amplifying said plurality of target nucleic acids in the sample in an amplification reaction that includes at least a two-step annealing/elongation temperature profile including a first step comprising said first temperature and a second step comprising said second temperature; (c) detecting, sequentially, over at least a portion of said annealing/elongation temperature profile (i) a first dectable signal during said first step, which results from annealing/elongation of the first primer and which corresponds to the signal emitted by said first label; (ii) a second detectable signal during said second step, which results from annealing/elongation of the first and second primers and which corresponds to the signal emitted by said first and said second label; and (iii) subtracting said first detectable signal from said second detectable signal to identify the detectable signal emitted by said second label; and (i) a first dectable signal during said first step, which results from annealing/elongation of the first primer and which corresponds to the signal emitted by said first label; (ii) a second detectable signal during said second step, which results from annealing/elongation of the first and second primers and which corresponds to the signal emitted by said first and said second label; and (iii) subtracting said first detectable signal from said second detectable signal to identify the detectable signal emitted by said second label; and (d) measuring a relative amount of each target nucleic acid in said plurality of target nucleic acids.