Protoplasts serve as a powerful system to study various plant physiological processes and to understand crucial signaling pathways within the plant system. The isolation of Arabidopsis protoplasts is a well-established technique and being utilized for wide range of assays. The method described herein includes the precise cutting of Arabidopsis leaves using extraction buffer containing cellulase and macerozyme. Further we present a polyethylene glycol [PEG]-mediated protoplast transformation method. Here, we have elucidated the methodology for isolation of protoplast for the AtSWEET-mediated sucrose uptake assay in control, and Pseudomonas syringe pv tomato DC3000 (Pst DC3000) treated leaves by utilizing GC/MS analysis. Our approach includes certain modifications to the previously published protoplast isolation technique, streamlining the process and providing a more accessible alternative to the highly specialized Xenopus oocyte uptake assay.