The Kv1.3 channels expressed by human T lymphocytes are emerging as important therapeutic targets. Peptides like agitoxin and margatoxin in scorpion venom and some non-peptide small molecules are known to inhibit this channel. Since such blockers cannot be used as drugs, pharma has a need to discover effective blockers. The major limiting factor for such development has been the lack of a reliable high-throughput screening (HTS) technology. A cell-based HTS assay for this target was developed in 96-well format to facilitate screening of many candidates. The assay incorporates rubidium ion as a tracer for potassium ion, which can be analyzed by the atomic absorption spectroscopy. The assay provided a Z' factor of 0.813 with more than a 4.5-fold window of detection. The two known blockers agitoxin and margatoxin gave a 50% inhibitory concentration (IC(50)) of 1.52 and 2 nM, respectively. These values are about five- and 2.8-fold higher than their IC(50) values obtained from patch clamp. Some non-peptide compounds like tamoxifen, nifedipine, and fluoxetine also inhibited the efflux through these channels, whereas astemizole and pimozide (potent human ether-a-go-go-related gene blockers) did not block Kv1.3 activity.