Hyperthermia has long been used in the treatment of cancer, through a variety of modalities, with temperatures ranging from fever-level (39 °C) to greater than 1,000 °C (electrocautery). The range of temperatures have very different effects, which is directly related to the temperature and treatment time (thermal dose) with high temperatures resulting in the tissue ablation and lower temperatures creating a variety of sublethal effects such as increased blood flow, accumulation of drugs, and immune stimulation. One of the more recent methods for delivering medical hyperthermia is magnetic nanoparticle therapy. This technique involves activating magnetic nanoparticles that can reside inside or outside of cells. The size and construct of the magnetic nanoparticles and the frequency and field strength of the magnetic field are major heating determinants. Using both in vitro and in vivo techniques and instrumentation, we have assembled a sophisticated process for delivering reproducible hyperthermia in large animal, small animal, and cell biology settings. This approach, using continuous, real time temperature monitoring in multiple locations, allows for the delivery of well-defined doses to the target tissue (tumor) while limiting non-target tissue heating. Precise control and monitoring of temperature allows for the accurate determination of the global quantitative hyperthermia standard: cumulative equivalent minutes at 43 °C (CEM43). Our system, which allows for a wide variety of temperatures, thermal doses, and biological effects, was developed through a combination of component acquisitions and inhouse engineering and biology. This system has been optimized in a manner that allows for the rapid conversion between ex vivo, in vitro, and in vivo situations. The goal of this protocol is to demonstrate how to design and implement an effective technique and system for delivering robust and accurate experimental in vitro and in vivo magnetic nanoparticle therapy (mNP) hyperthermia.