The objective of this study was to compare the performance of an immunoassay screening for synthetic cannabinoids with a newly developed confirmation method using liquid chromatography quadrupole time-of-flight mass spectrometry. The screening included metabolites from JWH-018, JWH-073, and AM-2201. The confirmation included metabolites from AM-2201, JWH-018, JWH-019, JWH-073, JWH-081, JWH-122, JWH-210, JWH-250, JWH-398, MAM-2201, RCS-4, and UR-144. The immunoassay was tested and found to have no cross-reactivity with UR-144 metabolites but considerable cross-reactivity with MAM-2201 and JWH-122 metabolites. Sensitivity and specificity for the immunoassay were evaluated with 87 authentic urine samples and found to be 87% and 82%, respectively. With a cutoff at 2 ng/ml, the confirmation showed 80 positive findings in 38 cases. The most common finding was JWH-122 5-OH-pentyl, followed by JWH-018 5-OH-pentyl. There were 9 findings of UR-144 metabolites and 3 of JWH-073 metabolites. In summary, the immunoassay performed well, presenting both high sensitivity and specificity for the synthetic cannabinoids present in the urine samples tested. The rapid exchange of one cannabinoid for another may pose problems for immunoassays as well as for confirmation methods. However, we consider time-of-flight mass spectrometry to be superior since new metabolites can be quickly included and identified.