Signal transduction is the process by which molecular signals are transmitted from the cell surface to its interior, resulting in functional changes inside the cell. B cell receptor (BCR) signaling is of crucial importance for B cells, as it regulates their differentiation, selection, survival, cellular activation and proliferation. Upon BCR engagement by antigen several protein kinases, lipases and linker molecules become phosphorylated. Phosphoflow cytometry (phosphoflow) is a flow cytometry-based method allowing for analysis of protein phosphorylation in single cells. Due to recent advances in methodology and antibody availability - together with the relatively easy quantification of phosphorylation - phosphoflow is increasingly and more commonly used, compared to classical western blot analysis. It can however be challenging to set-up a method that works for all targets of interest. Here, we present a step-by-step phosphoflow protocol allowing the evaluation of the phosphorylation status of signaling molecules in conjunction with extensive staining to identify various human and murine B cell subpopulations, as was previously published in the original paper by Rip et al. (2020). Next to a description of phosphoflow targets from the original paper, we provide directions on additional targets that play a pivotal role in BCR signaling. The step-by-step phosphoflow protocol is user-friendly and provides sensitive detection of phosphorylation of various BCR signaling molecules in human and murine B cell subpopulations.