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Critical step is sample extraction and preparation in analysis of biomolecules.Materials and techniques are improving for extracting nucleotides and nucleosides.How to analyze and extract the proteins in an exact lesion of diseased tissue.This review describes the recent developments in sample extraction and preparation techniques for mass spectrometric analysis of nucleotides, nucleosides, and proteins. Unique materials and techniques have been developed for highly selective extraction of nucleotides and nucleosides by solid-phase extraction strategies using various affinities. However, for proteins, the analysis of small-scale sections of diseased tissues (formalin-fixed, paraffin-embedded tissues) and the direct analysis of an exact lesion on the surface of diseased tissues (liquid extraction surface analysis) have become important advances in this field. In this review, we focus on the latest developments of these techniques and strategies.