Abstract Mass cytometry, which utilizes CyTOF® technology, is a single-cell analysis platform that uses metal-tagged antibodies. An advantage of CyTOF is its ability to resolve more than 40 parameters in a single panel without the need for compensation, making mass cytometry an ideal solution for routine enumeration of immune cells. Developing a robust and highly multiplexed assay requires an optimized panel and analysis pipeline, and the analysis of complex panels is time-consuming and difficult to interpret without expertise in immunology. The Maxpar® Direct Immune Profiling Assay™ coupled with Maxpar Pathsetter™ software is a sample-to-answer solution for human immune profiling using mass cytometry. The Maxpar Direct Immune Profiling Assay kit includes an optimized 30-marker panel contained in a lyophilized single-tube format, protocols for human whole blood and PBMC staining, and data acquisition instructions on a Helios™ system. Maxpar Pathsetter is an automated software that accepts FCS 3.0 files and reports cell counts, percentage calculations, and staining intensity. It also produces graphical elements such as histograms, dot plots, and a Cen-se’™ (t-SNE variant) graph for 36 immune cell populations. We present analytical validation data on repeatability and reproducibility for the assay and on the precision and accuracy of Maxpar Pathsetter software. We demonstrate comparative performance between the lyophilized panel and a liquid panel of the same antibodies. Coupling Maxpar Pathsetter software with the Maxpar Direct Immune Profiling Assay reduces variability in sample preparation and subjectivity in data analysis. It allows researchers to have a streamlined solution for broad immune profiling using mass cytometry.