This chapter describes techniques for the preparation of human spermatozoa for assisted conception. Traditional swim-up techniques depend on the intrinsic motility of human spermatozoa and have been used as the basis for new microfluidics devices that exploit the capacity of these cells for chemotaxis, rheotaxis or thermotaxis. Unfortunately, such self-migration strategies tend to lose effectiveness with pathological samples exhibiting poor levels of motility and/or diminished counts. With such compromised starting material, active selection of spermatozoa can be achieved by exploiting differences between sperm populations in their density or charge. Density gradient centrifugation selects spermatozoa with minimal residual cytoplasm that are highly motile and have good morphology, even though the induction of sperm DNA damage in certain patients may be problematical. Alternatively, methodologies based on differences in sperm charge, appear to hold promise for the rapid, efficient isolation of highly motile human spermatozoa that consistently exhibit low levels of DNA damage.