The confocal laser scanning microscope (CLSM) has become an essential tool for a wide range of biological and clinical studies and imaging applications. The major imaging modes of the CLSM include single and serial optical sections from thick specimens (100 µm), multiple wavelength images and three-dimensional reconstruction. Such images provide measurement of length, surface or volume of objects. Surprisingly, this advanced microscopic method has been very rarely used in anthropological research, although numerous clinical and basic science research projects showed its value to study, bone growth, bone micro-architecture and 3D bone morphometry. We present the basic principles and advantages of CLSM and outline practical aspects of specimen preparation, image collection and digital image processing specifically appropriate for historical bone and hair material. Copyright © 2009 John Wiley & Sons, Ltd.