In orthopaedic and dental surgery, bone grafts are used by surgeons as substitutes for the patient’s own bone. A bone graft provides support to the skeletal structure and stimulates the ingrowth of new bone. There is a growing demand for bone grafts due to an increasing world population and an ageing demographic. Tissue banks produce bone grafts originating from either human bone or animal bone. Bone grafts have been investigated profoundly. However, there is no literature that provides a standard protocol for the production process of bone grafts. The current production processes for bone grafts are inefficient because of many manual interferences by humans and consist of many complex production steps. This thesis aims to provide a bone processing protocol and investigates the first steps to developing an automated production line for bone processing. The automated production line must transform fresh donor bone to an end-product that is a clean and sterile allograft consisting of bone mineral and collagen. There is a hiatus in the knowledge about processing bone with multiple ultrasonic frequencies. This thesis provides new insights in processing bone with ultrasonic frequencies. Acoustic cavitation is produced by ultrasound and is used as a mechanical cleaning force. The literature review suggested that combining ultrasound frequencies is beneficial for cleaning. Low ultrasonic frequencies of 35 kHz have a better potential in removing large particles such as blood clots. High frequencies of 130 kHz are better for cleaning small particles such as small lipids and virus particles. This thesis researches the combination of multiple ultrasonic frequencies which should lead to optimal cleaning results. This thesis solves these research goals through an iterative process based on an extensive literature review, numerical modelling, testing and analysing cleaning procedures for bone samples. Human femoral heads were used as test samples. This thesis contributes new knowledge for cleaning bone with ultrasound and generates a new design for an automated process for cleaning bones.