A homogenous microparticle-based immunoassay has been developed for the detection of d-lysergic acid diethylamide (LSD) in human urine using the Online technology. This immunoassay is based on the principle of the kinetic interaction of microparticles in a solution where the drug content of the urine is directly proportional to the inhibition of the microparticle aggregation. Antibodies to LSD were obtained by immunizing goats with an LSD analogue derivatized through the indole nitrogen and conjugated to bovine thyroglobulin. The assay cutoff is 0.5 ng/mL LSD, and the clinical sensitivity for the detection of LSD and its metabolites in human urine samples is equivalent to the LSD Abuscreen RIA. Thirty-one samples previously screened LSD positive by Abuscreen RIA and confirmed by gas chromatography-mass spectrometry were analyzed by the Abuscreen OnLine LSD and Abuscreen LSD RIA assays. All thirty-one samples screened positive in the Abuscreen OnLine and Abuscreen RIA. OnLine's cross-reactivity to nor-LSD is greater than thirty-five percent; other structurally related compounds have similar cross-reactivity to that of the Abuscreen RIA. One thousand presumed negative urine samples were also analyzed; 992 (99.2%) of these gave negative results. The eight OnLine positive samples from this set were found to be negative by Abuscreen RIA. Typical qualitative within-run precision on the Hitachi 717 (at x = cutoff of 0.5 ng/mL; 0.5x, 1.0x, and 1.5x) was found to be less than 2.5%. Between-run precision was less than 3.0%.