In proteomics, there is a major challenge in how the functional significance of overexpressed proteins can be interpreted. This is particularly the case when examining proteins in cells or tissues. Here we have analyzed the physicochemical parameters, abundance level, half-life and degree of intrinsic disorder of proteins previously overexpressed in the yeast Saccharomyces cerevisiae. We also examined the interaction domains present and the manner in which overexpressed proteins are, or are not, associated with known complexes. We found a number of protein characteristics were strongly associated with deleterious phenotypes. These included protein abundance (where low-abundance proteins tend to be deleterious on overexpression), intrinsic disorder (where a striking association was seen between percent disorder and degree of deleterious effect), and the number of likely domain-domain interactions. Furthermore, we found a number of domain types, for example, DUF221 and the ubiquitin interaction motif, that were present predominantly in proteins that are deleterious on overexpression. Together, these results provide strong evidence that particular types of proteins are deleterious on overexpression whereas others are not. These factors can be considered in the interpretation of protein expression differences in proteomic experiments.