This book consists of 10 chapters that address current issues involving in vitro and in vivo biological assays. In vitro and in vivo bioassays are essential for the discovery and development of biologically-active agents. The techniques ensure that the discovered agent is worthy of a clinical study. Unfortunately, many gave little thought on bioassays’ choice at the discovery stage, thus creating false leads, low-quality, and non-reproducible data. Many discoveries from such poorly-considered bioassays fall short at the clinical stage and waste precious resources. This book deliberates on the issues in popular in vitro and in vivo bioassays for a better perspective of their limitations, hence ensuring accurate interpretation of findings. The book carries two themes: in vitro and in vivo bioactivity assays. Chapters 1 to 6 discuss prevalent in vitro bioassays—antiviral, cytotoxicity, scratch assay, and cellular biochemistry assays. Chapters 7 to 9 introduce in vivo studies in small animals and 3D culture in a dish. The book also presents special feature of in silico approach in Chapter 10, which offers a glimpse of computational power to support in vitro and in vivo biological findings. Studies discussed in this volume will benefit researchers in academia and the industry whose work might be focused on discovering the next bioactive agents.