An improved technique to generate high yields of relatively pure seminiferous tubule-enriched fractions from mouse testis by manual isolation is described. Our laboratory had previously developed an isolation method based on mild enzymatic digestion to separate individual constituents of each compartment of the testis, namely, the interstitial tissue and the seminiferous tubules. Although the method had the advantage of allowing the production of seminiferous tubule-enriched fractions in large amounts, we show here that this approach does not allow optimal preservation of the integrity of the proteins in the samples, in particular of the phosphorylated and/or glycosylated forms of the proteins. In an attempt to solve this problem, we developed a novel mechanical approach to generate interstitial tissue- and seminiferous tubule-enriched fractions that does not require the use of enzymatic digestion. This approach has the advantages of providing relatively pure seminiferous tubule-enriched fractions in large quantities and in a short amount of time. In addition, and more significantly, the approach allows a more faithful detection of the phosphorylated and glycosylated forms of the proteins.