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This paper discusses real-time sperm separation technology and the use of Wang tubes for isolating high-quality and fertile sperm. Several methods have been developed and used to prepare a better sperm specimen, including the conventional swim-up technique, Percoll discontinuous density gradient, Sephadex gel filtration, centrifugation, migration-sedimentation, albumin column, and Sperm-prep. However, none of these methods has the ability to isolate from the original ejaculum a large number of highly motile, morphologically normal, and normal-chromatin sperm, while retaining in an adequate volume of physiological medium, free of less-motile and nonmotile sperm, and free of microorganisms, cell debris, seminal plasma, leukocytes, and foreign bodies. These methods should be classified as incomplete techniques of sperm separation and are of only limited effectiveness in infertility treatment. They may also cause irreparable damage to the mother or fetus. Real-time sperm separation technology is based on the observations that nonpathological spermatozoa do not transfer organisms, that the motility pattern and swim-up capacity of pathological sperm are limited or disturbed, and that the movement of sperm differs from the passive motion of nonciliated microorganisms and the random active motion of ciliated microorganisms in terms of velocity and direction. Real-time sperm separation technology encompasses the Wang tube system, which has two main functions: to isolate organisms-free, normal-chromatin, morphologically normal, and highly motile spermatozoa that are suitable for use in infertility treatment, and to prevent sex-linked inherited diseases. The results of these evaluations indicate that real-time sperm separation technology is the most effective and precise method of isolating and decontaminating sperm to date.