Stomata, which are microscopic valves on the leaf surface formed by two guard cells (GC), play a critical role in the regulation of leaf water and gas exchange and, hence, determine plant adaptive potential. However, little data is available on GC biochemistry, protein abundance and gene expression, mainly due to technical difficulties and challenges in isolating sufficient amounts of high-quality pure GC. In the present study we applied some modifications to the mechanical isolation of guard-cell to generalise this method for diverse growth conditions as well as plant species. Epidermal peel fragments enriched in guard cells were mechanically isolated from quinoa, spinach and sugar beet leaves grown at two conditions (normal and salt stress). Multiple analysis was performed to confirm the suitability and superiority of the modified technique to the original method. At the first step, the viability and purity of GC-enriched epidermal fragments were assessed under the microscope. Then, the RNA integrity, gene expression, and 1D SDS-PAGE tests were performed to validate the suitability of this technique for omics studies. The data revealed a wide range of proteins as well as a high integrity of RNA extracted from guard cell samples. The expression level of several GC-specific genes and mesophyll-dominant genes were investigated using a comparative analysis of transcriptome datasets of GC and whole-leaf samples. We found that Rubisco and photosynthesis-related proteins such as chlorophyll a/b binding protein were substantially higher in the whole leaf compared with the GCs. More importantly, GC-specific genes such as OST1, SLAC1, MYB60, FAMA and HT1 were highly expressed in the GCs, confirming that our guard cell preparation was highly enriched in GC gene transcripts. Real-Time quantitative reverse transcription PCR further confirmed the efficacy of the GC isolation technique for exploring responses of GC to diverse types of stress at the molecular level.