The presence of serum in biological samples often negatively impacts the quality of in vitro assays. However, assays tolerant of serum are useful for assessing the in vivo availability of a small molecule for its target. Electrophysiology assays of ion channels are notoriously sensitive to serum because of their reliance on the interaction of the plasma membrane with a recording electrode. Here we investigate the tolerance of an automated electrophysiology assay for a voltage-gated potassium (K(V)) channel to serum and purified plasma proteins. The delayed rectifier channel, K(V)2.1, stably expressed in Chinese hamster ovary cells produces large, stable currents on the IonWorks Quattro platform (MDS Analytical Technologies, Sunnyvale, CA), making it an ideal test case. K(V)2.1 currents recorded on this platform are highly resistant to serum, allowing recordings in as high as 33% serum. Using a set of compounds related to the K(V) channel blocker, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone, we show that shifts in compound potency with whole serum or isolated serum proteins can be reliably measured with this assay. Importantly, this assay is also relatively insensitive to plasma, allowing the creation of a bioassay for inhibitors of K(V)2.1 channel activity. Here we show that such a bioassay can quantify the levels of the gating modifier, guangxitoxin-1E, in plasma samples from mice dosed with the peptide. This study demonstrates the utility of using an automated electrophysiology platform for measuring serum shifts and for bioassays of ion channel modulators.