Ustekinumab is an effective treatment for psoriasis, but response varies between patients. The formation of anti-drug antibodies (ADAs) may explain part of this variation by reducing the free ustekinumab level. Currently, published analyses of the clinical impact of ADAs are incomplete. In this observational cross-sectional multicenter study of 340 patients, we evaluated the impact of ADAs on ustekinumab level and clinical response as assessed by the PASI. Circulating ADA levels were measured using two assays: a drug-sensitive radioimmunoassay and a drug-tolerant ELISA. Circulating ustekinumab levels were measured using an ELISA. ADAs were detected in 3.8% (95% confidence interval [CI] = 3.2–4.2) and in 10.6% (95% CI = 7.9–13.9) of patients using the radioimmunoassay and drug-tolerant ELISA, respectively. At least 85% of the ADAs were neutralizing. Compared with patients negative for ADAs, ADA positivity in the radioimmunoassay and drug-tolerant ELISA were associated with lower median ustekinumab levels (−0.62 μg/ml [95% CI = −1.190 to −0.30] and −0.74 μg/ml [95% CI = −1.09 to −0.47], respectively) and higher absolute PASI (6.6 [95% CI = 3.0–9.9] and 1.9 [95% CI = 0.4–4.0], respectively). Absence of detectable ustekinumab regardless of ADA status correlated with poor clinical outcome (median sample PASI 10.1, 6.5 [95% CI = 3.9–8.8] compared with patients positive for ustekinumab). In conclusion, substantially reduced drug exposure resulting from ADAs formation is associated with impaired clinical response.