Objective: To develop a process for the detection of amphetamine, methamphetamine and ephedrine by enzymelinked immunosorbent assay (ELISA), based on the polyclonal antibody and heterology principle. Methods: The 3-aminopropyl (3AP) and 4-aminobutyl (4AB) derivatives of amphetamine (A), methamphetamine (M) and ephedrine (E) were chemically synthesized. They were used for the preparations of immunogens and hapten tracers. Direct competitive ELISA of matrix combinations of antisera and hapten tracers were performed using amphetamine, methamphetamine and ephedrine as the analytes. Only the competitive reactions with specified sensitivity and specificity are selected. Results: The study discovered three assay combinations that demonstrated concentration-dependent competition of analyte (single or multiple). They passed the confirmation test for the cut-off concentration and had no cross-reactivity with other amines or structured related compounds. The assay combinations of 4ABAAb with 3APA-PO and 4ABE-Ab with 3APM-PO were specific for the detection of amphetamine with ephedrine and methamphetamine with ephedrine, respectively. The third assay combination of 4ABE-Ab with 3APE-PO was highly specific to ephedrine with negligible cross-reactivity from other structure-related compounds. Direct competitive ELISA of 4ABE-Ab with 3APM-PO has been proven useful in field tests for the detection of methamphetamine in urine samples from Thai truck drivers suspected of drug abuse.