Amlodipine is a calcium channel antagonist of the dihydropyridine group. It is effective for treating hypertension, chronic stable angina, and vasospastic angina. However, it is difficult clinically to pinpoint the maximum dosage for antihypertensive activity of the drug without having parallel data on the plasma drug concentrations. The methods for assaying amlodipine are either gas chromatography with electron capture detector or liquid chromatography coupled with tandem mass spectrometry (or with an electrochemical detector), which needs tedious derivatization, and is expensive and time consuming. Therefore, in this study we developed an enzyme immunoassay for determining amlodipine in plasma. Anti-amlodipine antibodies were produced following immunization of bovine serum albumin-amlodipine conjugate. These specific antibodies were used in a competitive biotin-avidin-based enzyme-linked immunosorbent assay to measure amlodipine in plasma. Biotin was linked to the antibodies in order to enhance the sensitivity of the assay. The assay was specific for the free form of amlodipine with a detection limit of 0.1 ng/ml and the intra- and interassay coefficient of variation ranged from 1.6-10.2%. This immunoassay provides a sensitive, reliable, rapid, and accurate method for determination of amlodipine in plasma, which can be used in therapeutic drug monitoring pharmacokinetic studies and pharmaceutical analysis.