Abstract Protein Phosphatase 1 (PP1) is a major serine/threonine-phosphatase whose activity is dependent on its binding to regulatory subunits known as PP1 interacting proteins (PIPs), responsible for targeting PP1 to a specific cellular location, specifying its substrate or regulating its action. Today, more than 200 PIPs have been described involving PP1 in panoply of cellular mechanisms. Moreover, several PIPs have been identified that are tissue and event specific. In addition, the diversity of PP1/PIP complexes can further be achieved by the existence of several PP1 isoforms that can bind preferentially to a certain PIP. Thus, PP1/PIP complexes are highly specific for a particular function in the cell, and as such, they are excellent pharmacological targets. Hence, an in-depth survey was taken to identify specific PP1α PIPs in human brain by a high-throughput Yeast Two-Hybrid approach. Sixty-six proteins were recognized to bind PP1α, 39 being novel PIPs. A large protein interaction databases search was also performed to integrate with the results of the PP1α Human Brain Yeast Two-Hybrid and a total of 246 interactions were retrieved.