Matrix-assisted laser desorption/ionization(MALDI) mass spectrometry(MS) has been widely used for analysis of biological molecules, especially macromolecules such as proteins. However, MALDI MS has a problem in small molecule (less than 1 kDa) analysis because of the signal saturation by organic matrixes in the low mass region. In imaging MS (IMS), inhomogeneous surface formation due to the co-crystallization process by organic MALDI matrixes limits the spatial resolution of the mass spectral image. Therefore, to make laser desorption/ionization (LDI) MS more suitable for mass spectral profiling and imaging of small molecules directly from raw biological tissues, LDI MS protocols with various alternative assisting materials were developed and applied to many biological systems of interest.;Colloidal graphite was used as a matrix for IMS of small molecules for the first time and methodologies for analyses of small metabolites in rat brain tissues, fruits, and plant tissues were developed. With rat brain tissues, the signal enhancement for cerebroside species by colloidal graphite was observed and images of cerebrosides were successfully generated by IMS. In addition, separation of isobaric lipid ions was performed by imaging tandem MS. Directly from Arabidopsis flowers, flavonoids were successfully profiled and heterogeneous distribution of flavonoids in petals was observed for the first time by graphite-assisted LDI(GALDI) IMS.;Aqueous-based colloidal silver solution was also investigated as an alternative matrix for IMS application. For Arabidopsis thaliana wild-type and its cer2 mutant flowers, direct profiling and imaging of cuticular wax compounds by silver LDI MS and gas chromatography (GC) - MS were carried out for the first time and cuticular wax metabolites of them were compared for predicting the function of cer2 gene products. Results from silver LDI MS showed a good agreement with those from traditional GC-MS analysis and we propose that cer2 gene mutation mainly affects the conversion from C30 fatty acid to C29 alkane in wax biosynthesis pathway. We also applied silver LDI MS to probe cholesterol, which is the major component of lipid raft domains, from the Astrocyte cell monolayer. With colloidal silver, cholesterol was readily detected with high sensitivity and cholesterol level changes on cell monolayer by methyl beta-cyclodextrin treatment were successfully assayed by introducing relative intensity profiling. Results from more rapid, and simpler silver-LDI MS cholesterol assay method also showed a good agreement with those from traditional enzymatic fluorometry cholesterol assay method.