Ambient ionization MS has become very popular in analytical science and has now evolved as an effective analytical tool in metabolomics, biological tissue imaging, protein and small molecule drug analysis, where biological samples are probed in a rapid and direct fashion with minimal sample preparation at ambient conditions. However, certain inherent challenges continue to hinder the vibrant prospects of these methods for in situ analyses or to replace conventional methods in bioanalysis. This review provides an introduction to the field and its application in bioanalysis, with an emphasis on the most recent developments and applications. Furthermore, ongoing challenges or limitations related to quantitation, sensitivity, selectivity, instrumentation and mass range of these ambient methods will also be discussed.