The ability to experimentally perturb biological systems has traditionally been limited to static pre-programmed or operator-controlled protocols. In contrast, real-time control allows dynamic probing of biological systems with perturbations that are computed on-the-fly during experimentation. Real-time control applications for biological research are available; however, these systems are costly and often restrict the flexibility and customization of experimental protocols. The Real-Time eXperiment Interface (RTXI) is an open source software platform for achieving hard real-time data acquisition and closed-loop control in biological experiments while retaining the flexibility needed for experimental settings. RTXI has enabled users to implement complex custom closed-loop protocols in single cell, cell network, animal, and human electrophysiology studies. RTXI is also used as a free and open source, customizable electrophysiology platform in open-loop studies requiring online data acquisition, processing, and visualization. RTXI is easy to install, can be used with an extensive range of external experimentation and data acquisition hardware, and includes standard modules for implementing common electrophysiology protocols.