Nanoparticles (NPs) are being investigated widely for use in biomedical applications such as imaging, drug delivery, and cancer therapy due to their small size and readily tunable properties. The ability to accurately characterize NPs and monitor their spatial distributions is highly desirable for effective use of NPs and evaluation of their potential adverse environmental, health, and safety effects. In this dissertation, a simple, fast, and sensitive method based on laser desorption/ionization mass spectrometry (LDI-MS) to characterize and track NPs in biological systems has been developed. This method is especially well suited for characterizing core-shell structured NPs, such as quantum dots (QDs), gold NPs (AuNPs) and magnetic NPs, in a variety of sample types. The surface functionalities attached to the cores of the NPs can be qualitatively and quantitatively analyzed by LDI-MS with ultra-low sample consumption. A new molecular imaging technique has been developed based on the LDI-MS detection of NPs. Tissues from mice injected with NPs can be obtained and readily imaged to identify the location of NPs in these tissues. NPs can be selectively detected in a multiplexed fashion in tissues and the intra-organ distributions of same-sized NPs are directly linked to their surface functionality.