Among specialised “-omics” strategies developed to provide traceable biomolecular links at the cellular and systems level, peptidome analysis offers an information-rich and self-sufficient approach enabling the quantitative and systematic measurements of peptides in different biological media. Peptidome is usually referred to as an array of peptides present in a particular sampled pool or degradome of biomarkers (cells, plasma, cerebrospinal fluid). A clear distinction is made regarding the nature of the biomarkers, which are endogenous and are of low molecular weight, and can include peptidic factors, hormones and metabolic products. Exogenous peptides are of lesser emphasis in the literature, primarily due to molecular diagnostics approaches and nanoscale technologies that are developed in parallel and inter-relate with peptidomics at the point of mutual applications. Peptidomic profiles obtained for different conditions and diseases can vary greatly and require similarly varied tools, the development and optimisation of which are continuing from the traditional biochemical methods to advanced multidimensional separations and bioinformatics-empowered mass spectrometry. This report highlights mainstream tendencies in peptidome analysis exemplified by recent developments from the perspective of analytical tools and technologies