The present review deals with the recent developments in metabolome analysis to improve the diagnosis of diseases and the identification of biomarkers. A particular focus has been put on the application of the non-invasive and up-to-date microsampling and microextraction procedures for the targeted/untargeted metabolome analysis from low volume of samples. These approaches aim to enhance sensitivity for the study of highly bioactive, short-lived, and low-abundant metabolites from complex matrices, especially when access to high sample volumes is not possible. The main strengths and weaknesses of those modern approaches as well as robust solutions to meet the undeniable challenges in metabolomics field are also presented. Moreover, some attractive alternatives for the better extraction of hydrophilic metabolites are discussed. Besides, information are given related to method validation results (such as recovery, matrix effect, detection limit, reproducibility, stability, etc.) as well as perspectives of these modern procedures in the context of diseases understanding, diagnosis, and follow up.