Despite advances in intraoperative surgical imaging, reliable discrimination of critical tissue during surgery remains challenging. As a result, decisions with potentially life-changing consequences for patients are still based on the surgeon’s subjective visual assessment. Hyperspectral imaging (HSI) provides a promising solution for objective intraoperative tissue characterisation, with the advantages of being non-contact, non-ionising and non-invasive. However, while its potential to aid surgical decision-making has been investigated for a range of applications, to date no real-time intraoperative HSI (iHSI) system has been presented that follows critical design considerations to ensure a satisfactory integration into the surgical workflow. By establishing functional and technical requirements of an intraoperative system for surgery, we present an iHSI system design that allows for real-time wide-field HSI and responsive surgical guidance in a highly constrained operating theatre. Two systems exploiting state-of-the-art industrial HSI cameras, respectively using linescan and snapshot imaging technology, were designed and investigated by performing assessments against established design criteria and ex vivo tissue experiments. Finally, we report the use of our real-time iHSI system in a clinical feasibility case study as part of a spinal fusion surgery. Our results demonstrate seamless integration into existing surgical workflows.