A new generation of ONLINE assays has been developed that offers improved performance and enhanced ease of use. This family of assays is being applied to both the COBAS INTEGRA and Roche/Hitachi line of analyzers. The four ONLINE DAT II assays that were evaluated included cocaine (benzoylecgonine) (BE), methadone (MDN), opiates (OP), and tetrahydrocannabinol (THC). The BE assay has a dual cutoff (150/300 ng/mL) with a dynamic range from 0 to 5000 ng/mL. The MDN assay has a cutoff of 300 ng/mL with a dynamic range from 0 to 2000 ng/mL. The opiates assay has a 300 ng/mL cutoff with a 0 to 2000 ng/mL range and a 2000 ng/mL cutoff with a 0 to 8000 ng/mL range. The THC assay has 20, 50, and 100 ng/mL cutoffs with 0 to 100, 0 to 300, and 0 to 300 ng/mL dynamic ranges, respectively. The ranges of the intra-assay precision (coefficients of variation for n = 20) run in the semiquantitative mode are 2.3-7.5% for BE, 2.0-3.8% for MDN, 1.9-4.2% for OP, and 3.9-5.2% for THC. The intra-assay qualitative precision for all of the assays as calculated from absorbance values is generally higher than that of the intra-assay semiquantitative precision at the cutoff. The qualitative precision ranges between 0.4% and 3.1%. The standard curve stability defined for the COBAS INTEGRA systems for these reagents ranges from 35 to 68 days. The clinical sensitivity and specificity were compared to the OnLine generation I and CEDIA immunoassays, as well as gas chromatography-mass spectrometry (GC-MS). The results indicate that for each assay, the sensitivity and specificity were the same or greater when compared to the other two immunoassay technologies. The results of each assay also correlated very well (> 99%) when compared with GC-MS.