<jats:title>Abstract</jats:title><jats:sec><jats:title>Purpose</jats:title><jats:p>In a previous study, a new method was described using the sperm immobilization test (SIT) with computer‐aided sperm analysis (CASA). However, obtaining high‐quality sperm as needed was a known issue. Here, we compared the results of using frozen‐thawed sperm and fresh sperm for the SIT using the CASA method.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>For the frozen‐thawed preparation, 500 μL of condensed semen and 500 μL of Sperm Freeze were mixed in a cryovial and cryopreserved in liquid nitrogen. Density gradient centrifugation was used for the collection of motile sperm in both the fresh and frozen‐thawed sperm preparations. A total of 50 serum samples were prepared for both the fresh and frozen‐thawed sperm with each sample tested containing 10 μL of serum, 1 μL of either fresh or frozen motile sperm suspension, and 2 μL of complement. Sperm motilities were measured using CASA after a 1‐hour incubation period for both fresh and frozen‐thawed sperm.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Both fresh and frozen‐thawed sperm reacted similarly when exposed to serum containing sperm‐immobilizing antibodies asserting the use of frozen‐thawed sperm for the diagnosis of immunological infertility.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>These results suggest the possibility of using cryopreserved sperm for the SIT when fresh sperm is unavailable.</jats:p></jats:sec>