Enzyme inhibitors have been utilized as useful tools for elucidating the function and structure of specific enzymes and for cell biology studies. Recently, chemical screening from natural sources and compound libraries has led to the rapid discovery of enzyme inhibitors. To create more useful inhibitors with high enzyme selectivity, and molecular probes for analyzing the precise mode of actions for enzymes, synthetic approaches based on natural products and bio-molecules are considered to have an important role in medicinal chemistry and chemical biology. In this review, the “focused library approach” for the development of inhibitors and modulators for enzymes related to protein phosphorylations and de-phosphorylations was introduced. As protein kinase C modulators, we constructed a focused library with the conformationally-constrained 1,2-diacylglycerol (DAG) motif as the core structure. Among the synthesized compounds, we found some characteristic molecules with different binding affinity to the C1 domain and activation ability for PKCα. As inhibitors for the dual-specificity protein phosphatase VHR, the neutral phosphate-mimicking core structure was designed based on natural product RK-682. Among the derivatives of the constructed focused library, including the neutral core structure stated above, we found the selective inhibitor for VHR, which showed cell cycle arresting activity for NIH3T3 cells and inhibitory activity for the de-phosphorylation of ERK and JNK.