Spermatozoa with deteriorated plasma membranes can be separated by magnetic-activated cell sorting (MACS) after binding superparamagnetic annexin V-conjugated microbeads (ANMBs) to membrane phosphatidylserine (PS). Semen samples from 15 donors and 25 infertile patients were divided into 2 spermatozoal fractions by annexin V-MACS. Activated caspases (aCPs), which mediate degradations of cell quality, were determined by CaspaTag in the 2 subpopulations. Spermatozoa from donors showed lower levels of bound annexin V (3.6% Ϯ 0.5% vs 11.9% Ϯ 1.1%; P Ͻ .01) and aCPs (21.8% Ϯ 2.6% vs 43.2% Ϯ 2.1%; P Ͻ .01) than did spermatozoa from infertile patients. MACS resulted in a decrease of spermatozoa with aCPs from 21.8% Ϯ 2.6% (before separation) to 9.2% Ϯ 1.4%(in the ANMB-negative fraction) in donors and from 43.2% Ϯ 2.1% to 18.8% Ϯ 2.6% in infertile patients (mean Ϯ SEM; P Ͻ .01). Separation effects of the MACS technique were confirmed with flow cytometry using anti-annexin V antibodies and with electron microscopy. ANMB-MACS removes spermatozoa with PS-bound annexin V and produces a higher quality spermatozoal fraction. Spermatozoa with a deteriorated membrane are characterized by an increase in aCPs. A higher percentage of spermatozoa with ANMBs bound to PS and with aCPs were found in infertile patients.