Targeting the PI3K isoform p110δ against B cell malignancies is at the mainstay of PI3K inhibitor (PI3Ki) development. Therefore, we generated isogenic cell lines, which express wild type or mutant p110δ, for assessing the potency, isoform-selectivity and molecular interactions of various PI3Ki chemotypes. The affinity pocket mutation I777M maintains p110δ activity in the presence of idelalisib, as indicated by intracellular AKT phosphorylation, and rescues cell functions such as p110δ-dependent cell viability. Resistance owing to this substitution consistently affects the potency of p110δ-selective in contrast to most multi-targeted PI3Ki, thus distinguishing usually propeller-shaped and typically flat molecules. Accordingly, molecular dynamics simulations indicate that the I777M substitution disturbs conformational flexibility in the specificity or affinity pockets of p110δ that is necessary for binding idelalisib or ZSTK474, but not copanlisib. In summary, cell-based and molecular exploration provide comparative characterization of currently developed PI3Ki and structural insights for future PI3Ki design.