Cryopreservation of stallion spermatozoa causes significant cell damage, initially from structural damage due to freeze-thaw cycles, followed by the generation of reactive oxygen species (ROS). To maximize the longevity of spermatozoa post-thaw, it is essential to isolate a sub-population of viable, low ROS-producing spermatozoa. The Felix™ system (Memphasys, Australia) is a commercial electrophoretic sperm isolation device capable of isolating highly motile spermatozoa with low levels of DNA damage. The aim of this study was to ascertain whether the quality of frozen-thawed spermatozoa could be improved by isolating viable sperm prior to storage. Stallion spermatozoa (n = 6 ejaculates) were frozen in lactose-EDTA cryodiluent containing 20% egg yolk and 3% dimethylformamide, thawed, and either washed via centrifugation (control), or viable sperm were isolated with either the Felix™ system or via single-layer colloidal centrifugation through Equipure™, followed by resuspension into SpermSafe™ medium for up to 24 h of storage at 17 °C. Following 1 and 24 h of storage, Equipure™ and Felix™-isolated cells exhibited higher total and progressive motilities compared to the control (CASA; P ≤ 0.05). Additionally, these isolated cells had less DNA fragmentation (Halo assay; P ≤ 0.05). Flow cytometry was used to assess cell viability (far-red LIVE/DEAD™), mitochondrial ROS generation (MitoSox Red™), and lipid peroxidation (4-hydroxynonenal adduct formation). Isolated cells had higher viability (P ≤ 0.05) and lower levels of mitochondrial ROS and lipid peroxidation (P ≤ 0.05) in cells isolated with both Equipure™ and Felix™. However, the concentration of samples isolated with the Felix™ system was lower than Equipure™ isolated samples (P ≤ 0.05). This indicates that while Felix™ is more time-efficient (6 min protocol) compared to Equipure™ (20 min protocol) and produces samples of similar quality, the yield is only viable for applications such as IVF or ICSI.