Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.