Solid phase microextraction (SPME) is a rapid, well-established, and solvent-free sample preparation technique. It has usually been hyphenated with GC-MS or LC-MS/MS to separate and to detect enriched analytes. In recent years, ambient ionization techniques have been developed fast. This has allowed MS to be used for direct and straightforward SPME analysis of complex samples, dismissing the need for chromatographic separation. SPME sampling disturbs systems to a minimum—it removes (extracts) only small fractions of analytes. Therefore, in vivo analysis is a special application area where SPME is gaining ground. This review summarizes state-of-the-art in vivo SPME for bioanalysis (in human, rats, fishes, rhesus macaque, frog, and living cattle), including biocompatible SPME coatings, sampling approaches for in vivo SPME (skin, exhaled breath, saliva, blood, muscle, and brain sampling), analytical/detection systems, and calibration methods. This review also discusses in vivo SPME applications (pharmacokinetic studies, exposure, biomarkers, diagnosis, doping control, lipidomics, drug analysis, cosmetic dermatology, bioaccumulation, biomonitoring, environmental pollutants, and therapeutic drug monitoring) and future trends in in vivo SPME bioanalysis. •State-of-the-art in vivo SPME for bioanalysis.•In vivo SPME applications.•Future trends in in vivo SPME bioanalysis.