Introduction of fluorescence-based Real-Time PCR (RT-PCR) also called qPCR is increasingly used to detect multiple pathogens simultaneously and rapidly by gene expression analysis of PCR amplification data. Real-time PCR data are analyzed after setting an arbitrary threshold that must intersect the signal curve in its exponential phase. The point at which the curve crosses the threshold is called CT ( Cycle Threshold). This simple and arbitrary value however is not an elegant definition of CT value sometimes leads to conclusions that are either false positive or negative. Therefore, the purpose of this paper is to present a stable and consistent alternative approach for the definition and determination of CT value that leads to near zero false positives and negatives.