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Single cells offer a simplified model for investigating complex mechanisms such as cell–cell adhesion. Protoplasts, plant cells without cell walls (CWs), have been instrumental in plant research, industrial applications, and breeding. However, because of the absence of a CW, protoplasts are not considered “true” plant cells, making them less relevant for biophysical studies. Current protocols for CW recovery in protoplasts vary widely among laboratories and starting materials, requiring lab-specific optimizations that often depend on expert knowledge and qualitative assessments. To address this, we have developed a user-friendly streamlined workflow, the Q-Warg pipeline, which enables quantitative comparison of various conditions for CW recovery post-protoplasting. This pipeline employs fluorescence imaging and tailored processing to measure parameters such as morphometry, cell viability, and CW staining intensity. Using this approach, we optimized culture conditions to obtain single plant cells (SPCs) with recovered CWs. Additionally, we demonstrated the robustness and versatility of the workflow by quantifying different fluorescent signals in protoplast suspensions. Overall, the Q-Warg pipeline provides a widely accessible and user-friendly solution for robust and unbiased characterization of protoplasts culture. The quantitative data generated by the pipeline will be useful in the future to decipher the mechanisms regulating protoplast viability and regeneration.