AbstractWe describe here a simple and efficient antibody titration approach for cell‐surface markers and intracellular cell signaling targets for mass cytometry. The iterative approach builds upon a well‐characterized backbone panel of antibodies and analysis using bioinformatic tools such as SPADE. Healthy peripheral blood and bone marrow cells are stained with a pre‐optimized “backbone” antibody panel in addition to the progressively diluted (titrated) antibodies. Clustering based on the backbone panel enables the titration of each antibody against a rich hematopoietic background and assures that nonspecific binding and signal spillover can be quantified accurately. Using a slightly expanded backbone panel, antibodies quantifying changes in transcription factors and phosphorylated antigens are titrated on ex vivo stimulated cells to optimize sensitivity and evaluate baseline expression. Based on this information, complex panels of antibodies can be thoroughly optimized for use on healthy whole blood and bone marrow and are easily adaptable to the investigation of samples from for example clinical studies. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.