The expansion of gene families during evolution, which can generate functional overlap or specialization among their members, is a characteristic feature of signalling pathways in complex organisms. For example, families of transcriptional activators and repressors mediate responses to the plant hormone auxin. Although these regulators were identified more than 20 years ago, their overlapping functions and compensating negative feedbacks have hampered their functional analyses. Studies using loss-of-function approaches in basal land plants and gain-of-function approaches in angiosperms have in part overcome these issues but have still left an incomplete understanding. Here, we propose that renewed emphasis on genetic analysis of multiple mutants and species will shed light on the role of gene families in auxin response. Combining loss-of-function mutations in auxin-response activators and repressors can unravel complex outputs enabled by expanded gene families, such as fine-tuned developmental outcomes and robustness. Similar approaches and concepts may help to analyse other regulatory pathways whose components are also encoded by large gene families.