Background: This study aimed to select high-quality spermatozoa by fresh domestic feline semen using sperm separation by magnetic activation (MASS), compared to density gradient centrifugation (DGC), and to evaluate cell quality after selection. Methods: Semen was collected from ten domestic felines by pharmacological sampling using dexmedetomidine and ketamine followed by urethral catheterization. The following parameters were analyzed: motility (computer assisted semen analysis), concentration (Neubauer chamber), semen morphology (humid chamber), and supravital test (eosin/nigrosine staining). In DGC, 20 × 106 spermatozoa were used in a gradient of Percoll at 90% and 45%, centrifuged at 900 g for 5 min, and the pellet was diluted in HEPES buffer. In MASS, 10 × 106 spermatozoa were diluted in HEPES buffer, centrifuged at 300 g for 10 min. The pellet was then resuspended in HEPES buffer with nanoparticles bound to annexin V, incubated for 15 min, and then filtered in the magnetic separation column. Results: The total abnormalities in the fresh semen were 47.9 ± 4.47%, with DGC and MASS being effective in reducing sperm abnormalities by 15.4 ± 0.95% and 29.80 ± 4.90%, respectively. Conclusions: This nanotechnological method is efficient in producing high-quality semen samples for assisted reproduction.