Synthetic analogues of natural oligonucleotides known as locked nucleic acids (LNAs) offer superior nuclease resistance and cytocompatibility for numerous scenarios ranging from in vitro detection to intracellular imaging of nucleic acids. While recognized as stronger hybridization partners than equivalent DNA residues, quantitative analysis of LNA hybridization activity is lacking, especially with respect to competitive displacement of the original hybridization partner by another oligonucleotide. In the current study, we perform in situ measurements of toehold-mediated competitive displacement of soluble, fluorescently labeled primary targets from probe strands immobilized on microspheres using high throughput flow cytometry. Both LNA-DNA hybrid sequences and pure DNA sequences are employed as the immobilized strands, as soluble, fluorescently labeled 9-base-long primary targets, and as unlabeled 15-base-long secondary or competitive targets. In addition to comparing chemically substituted and unsubstituted sequences, we explore the effects of mismatched primary targets and the location of the toehold segment within the primary duplexes on the resulting displacement profiles. The primary duplex or double-stranded probe (dsprobe) systems implemented here exhibited varying responses to unlabeled secondary targets ranging from surprisingly modest primary target displacement activity despite the presence of a six base-long nucleotide toehold segment at the dsprobe free end to distinctive displacement profiles sensitive to LNA substitutions and the placement of the toehold segment closer to the microsphere surface.