Taq DNA polymerase contains a polymerase domain for synthesizing new DNA strands and a 5'-nuclease domain for cleaving damaged DNA strands or RNA primers. Both of these domains play key roles in nucleic acid amplification and detection, especially in fluorogenic probe-based, real-time PCR. However, the 5'-nuclease activity is substrate dependent and its consequences remain largely unexplored, except for its role in 5'-nuclease-based TaqMan assays. Using both kinetic studies and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), we comprehensively examined the 5'-nuclease activity of Taq DNA polymerase on fluorogenic double-stranded probes of varied structures. We observed that double-stranded probes with destabilized 5'-terminal could be hydrolyzed, and the major cleavage was the removal of the 5'-terminal fluorophore-labeled nucleotide. These observations can serve as guidance for better design of double-stranded probes with reduced or no interfering background for real-time PCR detection.