Microtubule gliding assays provide a unique mechanism for molecular detection in which binding of analytes to the microtubule lattice reduces the microtubule gliding speed. The reduction in the gliding speed correlates with the density of the bound analytes, enabling its quantification. Although promising, this technique is still in the proof-of-concept stage. Improving the sensitivity and limit of detection of the assay could make the technique comparable to that of advanced molecular detection methods. This study demonstrates that reducing the ionic strength of the buffer increases the sensitivity of the assay by enhancing the interactions between kinesin and microtubules. When using a low ionic strength buffer (BRB10) compared with a standard buffer (BRB80), we observed a more pronounced reduction in microtubule gliding speed in the presence of analytes, improving the detection limit. Therefore, this approach offers a simple and scalable way to improve the sensitivity of motor-based detection assays.