Hydrogenated detonation nanodiamonds are of great interest for emerging applications in areas from biology and medicine to lubrication. Here, we compare the two main hydrogenation techniques—annealing in hydrogen and plasma-assisted hydrogenation—for the creation of detonation nanodiamonds with a hydrogen terminated surface from the same starting material. Synchrotron-based soft X-ray spectroscopy, infrared absorption spectroscopy, and electron energy loss spectroscopy were employed to quantify diamond and non-diamond carbon contents and determine the surface chemistries of all samples. Dynamic light scattering was used to study the particles’ colloidal properties in water. For the first time, steady-state and time-resolved fluorescence spectroscopy analysis at temperatures from room temperature down to 10 K was performed to investigate the particles’ fluorescence properties. Our results show that both hydrogenation techniques produce hydrogenated detonation nanodiamonds with overall similar physico-chemical and fluorescence properties.