Despite the recent advances in parallel protein-based analyses the proportion of the protein composition of any specific tissue or organism that is currently being analyzed is still unknown. The ultimate aim of proteomics is to characterize all of the proteins in a biological system under study, but much has been gained from knowledge of smaller subsets of the proteome. Therefore, while techniques and instrumentation are being improved to increase the sensitivity of analysis, it is just as important to answer the question of what depth of analysis is required for reasonable conclusions to be reached. The questions to be answered and the resulting depth of analysis required will vary depending upon whether the understanding is required for diagnostic markers, therapeutic targets or biological systems. The issues associated with increasing the depth of analysis of proteins in the context of these areas will be discussed. However, it should be noted that merely increasing the amount of data acquired will not necessarily increase the amount of knowledge of a particular system and as such careful implementation of proteomic methods is required to advance these fields of research.