Small molecule parenterals have often been formulated as solutions or suspensions in non-aqueous conditions, however, this technology has not found widespread use in the formulation of macromolecules. Formulation of proteins and peptides has primarily been achieved through aqueous solutions or reconstituted lyophilized cakes. The incorporation of non-aqueous techniques has been limited by the lack of general applicability. For example, prediction of solubility, chemical stability, conformational stability (unfolding/denaturation processes), and activity can be difficult. Therefore, macromolecule non-aqueous preformulation work must be performed on a case by case basis. In addition, only a few solvents are pharmaceutically acceptable. This article reviews the characterization of proteins and peptides in a variety of non-aqueous or co-solvent conditions (both acceptable and unacceptable for pharmaceutical applications), and discusses the applicability of non-aqueous conditions for increasing solubility , stability and activity.