Introduction Biological fluids are routine samples for diagnostic testing and monitoring. Blood samples are typically measured because of their moderate invasive collection and high information content on health and disease. Several body fluids, such as cerebrospinal fluid (CSF), are also studied and suited to specific pathologies. Over the last two decades, proteomics has quested to identify protein biomarkers but with limited success. Recent technologies and refined pipelines have accelerated the profiling of human biological fluids. Areas covered We review proteomic technologies for the identification of biomarkers. These are based on antibodies/aptamers arrays or mass spectrometry (MS), but new ones are emerging. Advances in scalability and throughput have allowed to better design studies and cope with the limited sample size that has until now prevailed due to technological constraints. With these enablers, plasma/serum, CSF, saliva, tears, urine, and milk proteomes have been further profiled; we provide a non-exhaustive picture of some recent highlights (mainly covering literature from the last 5 years in the Scopus database) using MS-based proteomics. Expert opinion While proteomics has been in the shadow of genomics for years, proteomic tools and methodologies have reached certain maturity. They are now better suited to discover innovative and robust biofluid biomarkers.