129Xe dissolution dynamic nuclear polarization (DNP) is a controversial topic. The gold standard technique for hyperpolarized xenon magnetic resonance imaging (MRI) is spin exchange optical pumping, which received FDA approval in 2022. Nevertheless, the versatility of DNP for enhancing the signal of any NMR active nucleus might provide new perspectives for hyperpolarized 129Xe NMR/MRI. Initial publications about 129Xe DNP underlined the increased complexity in the sample preparation and lower polarization levels when compared to more conventional 13C-labeled molecules, at same experimental conditions, despite very close gyromagnetic ratios. Herein, we introduce, using a Custom Fluid Path system, a user-friendly and very robust sample preparation method. Moreover, investigating the radical properties at real DNP conditions by means of LOngitudinal Detected Electron Spin Resonance, we discovered a dramatic shortening of the electron spin longitudinal relaxation time (T1e) of nitroxyl radicals in xenon DNP samples’ matrices, with respect to more commonly used water:glycerol ones. Mitigating those challenges through microwave frequency modulation, we achieved over 20% 129Xe polarization without employing any deuterated solvent.