Extracellular acidification or alkalization is a common response to many plant-signaling peptides and microbial elicitors. This may be a result of peptide-mediated regulation of plasma membrane-localized ion transporters, such as the plasma membrane H⁺-ATPase. Early responses to some signaling peptides can therefore be analyzed by assaying H⁺-pumping across the plasma membrane. We describe a set-up suited for the purification of plasma membranes by aqueous two-phase partitioning from a small sample of Arabidopsis seedlings. Seedlings are grown in a liquid culture, suited for the analysis of in vivo peptide treatment. Additionally, we describe how to measure the H⁺-pumping activity of the plasma membrane H⁺-ATPase using the fluorescent probe ACMA.