Abstract: Apoplast washing fluid (AWF) contains minerals, metabolites, and proteins that plays an important role in plant growth and development, as well as provides biotic and abiotic stress resistance. AWF extraction is the basis of exploring the function of AWF constituents. It is generally performed via vacuum infiltration-centrifugation technique; which varies in processes and detailed parameters depending on the plan species, organs, and culture conditions. Hydro-cultured cotton seedlings were used to investigate AWF separation processes and parameters suitable for cotton root and leaf development, and further improve methods for cotton root or leaf AWF separation. Compared with traditionally split sampling (i.e., splitting samples into segments or pieces), sampling a complete unit was simple and significantly decreased the ratio of malate dehydrogenase (MDH) activity in AWF to symplast washing fluid (SWF), which usually is used to affirm the degree of AWF substances polluted by SWF; indicating that AWF components would better to examine. Furthermore, the fresh weight increments and the AWF diluting factor after vacuum infiltration of the roots had no significant change, but significantly increased in the leaves. This indicates that vacuum infiltration is only essential for leaves, with a vacuum strength/time at -60 kPa/1 min, and about 110 s recovery from vacuum to normal atmospheric pressure. Leaf areas with dark color increased with vacuum intensity or time, which could be used as a simple indicator for determining the suitability for AWF separation. Finally, comprehensive analyses of the AWF volume, soluble protein content ratio and MDH activity ratio of AWF to SWF indicated that the suitable centrifuge forge/time was 800×g/10-20 min for the root, and 400×g/5 min for the leaf. This refined and optimized method will lay down the foundation for efficient study of AWF components such as the accuracy and reliability of proteomics and metabolomics. The approach towards establishing this method should allow it to be generally applicable to other plants.