This session introduces basic concepts of imaging-based high-throughput screening (HTS) and high-throughput profiling assay development. Imaging-based HTS assays are designed to evaluate a discrete cellular process and produce a single, or small number of quantitative outputs. In contrast, imaging-based HTP assays measure dozens to thousands of features and provide highly multiplexed quantitative outputs. Either type of approach may be used to evaluate the effects of chemicals or other perturbagens on cellular biology. Topics for this session include (but are not limited to) considerations for model selection, endpoint selection, imaging assay design, identification and use of positive control and reference treatments, methods for evaluating assay dynamic range and approaches for evaluating assay reproducibility. Examples of HCS assays for a variety of biological processes including nuclear receptor activation, oxidative stress, apoptosis, neurite outgrowth and others will be explored. In addition, this presentation will explore the basic concepts of machine learning classification in the context of HTP assays such as Cell Painting. Attendees will gain a basic foundational knowledge of guiding principles underlying the development of imaging-based HTS and HTP assays. The views expressed in this presentation are those of the author and do not necessarily reflect USEPA policy.