Radio frequency (RF) biosensors are an expanding field of interest because of the ability to design noninvasive, label-free, low-production-cost sensing devices. Previous works identified the need for smaller experimental devices, requiring nanoliter to milliliter sampling volumes and increased capability of repeatable and sensitive measurement capability. The following work aims to verify a millimeter-sized, microstrip transmission line biosensor design with a microliter well operating on a broadband radio frequency range of 1.0–17.0 GHz. Three successive experiments were performed to provide evidence for (1) repeatability of measurements after loading/unloading the well, (2) sensitivity of measurement sets, and (3) methodology verification. Materials under test (MUTs) loaded into the well included deionized water, Tris-EDTA buffer, and lambda DNA. S-parameters were measured to determine interaction levels between the radio frequencies and MUTs during the broadband sweep. MUTs increasing in concentration were repeatably detected and demonstrated high measurement sensitivity, with the highest error value observed being 0.36%. Comparing Tris-EDTA buffer versus lambda DNA suspended in Tris-EDTA buffer suggests that introducing lambda DNA into the Tris-EDTA buffer repeatably alters S-parameters. The innovative aspect of this biosensor is that it can measure interactions of electromagnetic energy and MUTs in microliter quantities with high repeatability and sensitivity.