Calixarenes, macrocyclic compounds of phenolic units linked by methylene groups at the 2,6-positions, present some of the requirements to serve as platforms for the design and synthesis of biological active compounds. They are also interesting host molecules for chemical biology study purposes. Their basic molecular scaffold has potential ability for molecule recognition; it is promptly synthesized in large amounts, and might be easily modified for maximizing molecular interactions toward relevant guest molecules. Calixarenes present well-defined conformational properties and cavities with molecular dimensions that enable to encapsulate guest drugs. Calixarenes have been shown to have antiviral, antibacterial, antifungal, and anticancer activities (including HIV as target). We provide here an overview of the use of calixarenes either as new chemical entity of distinct biological activities or as host for bioactive guest molecules. The importance of calixarenes for drugs development is discussed. The use of Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) techniques for the study of calixarenes as biological molecule hosts is also described.