To achieve a cell-mimetic reaction environment, we fabricated and tested quartz microchambers for conducting protein synthesis using an in vitro transcription and translation system, the PURE system. By introducing a glass microchamber and blocking the surface of the chamber with amino acids, the concentration of the synthesized marker protein (green fluorescent protein, GFP) was significantly improved compared to that in the poly(dimethylsiloxane) (PDMS) microchamber. The concentration was below the detection limit in the PDMS microchambers, whereas the glass microchambers yielded 700 nM GFP, representing 41% of the bulk reaction. There was no detectable difference when the GFP synthesis was performed in microchambers with sizes ranging from 40 fL to 7 pL, indicating that the present microchamber system can serve as a cell-sized test tube with a variable reaction volume. Finally, we demonstrated that two different proteins, GFP and β-galactosidase, can be expressed from single genes in our experimental setup. Quantized and distinctive signals from proteins synthesized from 0, 1, or 2 copies of genes were obtained. The microchamber presented here can be utilized not only to study the effects of compartment volume on protein synthesis but also for the comprehensive analysis of complex biochemical reactions in cell-mimetic environments.