During the past several years, mass spectrometry has made great progress in the analytical field of biological molecules. The progress has been mainly achieved with the development of new soft ionization techniques, such as plasma desorption, laser desorption, molecular secondary ion mass spectrometry, and fast atom bombardment mass spectrometry, and with the development of a big mass spectrometer. It has been demonstrated that polar nonvolatile big bioorganic molecules can be ionized without dissociation by soft ionization methods, which are called desorption ionization. The soft ionization methods and recent applications are overviewed. Newly developed thermospray ionization seems to be obtaining a reputation as a suitable ionization method for LC/MS and several applications have been achieved more recently. The application of tandem mass spectrometry (MS/MS) to structure analysis or mixture analysis of bioorganic samples has started recently, while it was widely used as a method for analyzing the ion structure of small molecules. These combination techniques of using a mass spectrometer with a liquid chromatograph or another mass spectrometer are promising methods for mixture analysis and they are also reviewed here.