Methods for determining nucleic acid fragmentation status are provided. A nucleic acid of interest in a reaction mixture is contacted with two or more different probes complementary to sites separated by a point of potential fragmentation. The probes each comprise a detectable marker. The nucleic acid of interest is flowed into a detection region, where two or more coincident detectable marker signals from the probes are detected. Fragmentation status of the nucleic acid of interest is determined, coincident detection of signals from two or more of the different probes indicating the nucleic acid of interest is not fragmented between the probes.