Mass spectrometry, widely recognized for its high speed, specificity and sensitivity as well as applicability in the analysis of a broad range of samples, is playing an even-increasingly important role in a broad range of applications. The objectives of the projects described in this thesis research are to develop novel mass spectrometric methodologies for in situ detection of hazardous chemical and biological agents. The first project described is the development and characterization of novel linear ion trap mass analyzers based on the rectilinear geometry. Such mass analyzers consist of either four or six planar electrode and employ pure RF voltages (or with supplementary DC voltages) for two dimensional ion trapping. The structure simplicity makes these devices particularly significant for the development of miniaturized ion trap mass spectrometers. The second project described is the discovery of new ion/molecule reactions and the associated implementation via ambient ionization techniques desorption atmospheric pressure chemical ionization (DAPCI) and desorption electrospray ionization (DESI), for the specificity enhancement in in situ detection of nitroaromatic explosives and organophosphonates. Such additional specificity is especially valuable for in situ chemical hazard detection since complex mixtures are necessarily examined. The third project described is the application of DESI to profile fresh intact microorganisms. This study demonstrates the possibility of performing in situ identification, including sub-species differentiation of microbiological agents by using DESI-MS. The lack of sample treatment and the known sensitivity of the DESI experiment represent progress toward rapid in situ mass spectrometric identification of biological threats. Future directions should include integration of ambient ionizations to miniaturized mass spectrometers, to produce a versatile portable device for in situ detection of chemical and biological threats