Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient ionization technique which application in forensic chemistry appears to be very promising. The technique enables rapid in situ analyses without the need of elaborate sample preparation. Nowadays, many DESI-MS applications in forensics, medical diagnostics and food control have been already reported in the literature. The study summarizes the latest DESI-MS forensic applications in the analysis of biological fluids and tissues, i.e., fingerprints, skin, urine and blood, in which the interest has grown during the recent years. In the publication presented below, the use of blood samples in dried blood spots is also included. The analysis reported covers mainly the determination of medicines and illicit drugs content in biological fluids and tissues, where great DESI-MS potential in screening and drugs identification was recognized. Furthermore, the examples of explosives and condoms components detection are also present. Abbreviations ACN Acetonitrile DBS Dried blood spots DESI-MS Desorption electrospray ionization mass spectrometry DMF N,N-dimethylformamide EtOH Ethanol GC-MS Gas chromatography-mass spectrometry GSR Gunshot residues LC-MS/MS Liquid chromatography tandem mass spectrometry LOD Limit of detection LOQ Limit of quantification MeOH Methanol OTC drugs Over-the-counter drugs PMMA Poly(methyl methacrylate) PTFE Polytetrafluoroethylene RDX Trinitrohexahydro-1,3,5-triazine TFME Thin-film microextraction TLME Thin liquid membrane extraction TLC Thin-layer chromatography TNT 2,4,6-Trinitrotoluene QuEChERS extraction Quick, easy, cheap, effective, rugged, and safe extraction