Precise control of biological processes by the application of small molecules can increase the safety and efficiency of therapies. Adverse side effects of small molecule signals and/or immunogenicity of regulatory domains hinder their biomedical utility. Here, we designed small molecule-responsive switches, based on the conditional reassembly of human antibody variable fragments, called Fv-CID switches. The principle was validated using high-affinity antibodies against nicotine and β-estradiol to construct chemically responsive transcription factors. Further, we developed an Fv-CID switch responsive to bio-inert, clinically approved compound fluorescein, which was used to control the activity of chimeric antigen receptor (CAR) T cells and bispecific T cell engagers (BiTEs) in vivo. This study provides a framework to regulate the expression of endogenous genes, combine multiple chemical signals, and regulate T cell-based immunotherapy in an animal cancer model using a clinically approved small molecule regulator that could be customized for regulating therapeutic proteins or cells.