Adipose tissue (AT) is no longer considered merely as insulation or padding for human organs. It is an endocrine organ in its own right, which includes composite cells with the ability to differentiate into multiple cell lines. In fact, there is increasing evidence to support the theory that the causation of obesity and its associated metabolic disorders originate at the cellular or tissue level. Adipocyte dysfunction and chronic inflammatory states are able to modulate triglyceride storage and mobilization directly through cytokine and adipokine release. Significant variability exists between adipocyte isolation and culture techniques which subsequently can impact experimental results. We aim to explain the importance of controlling these variables, to assist tailoring methodological choice towards the investigational outcomes, and modifications of the techniques used accordingly. The techniques described in this chapter yield cell and adipose tissue which can be utilised in many different ways, including adipose tissue stem cells for differentiation, DNA analysis, RT-PCR, immunohistochemistry, lipolysis, glucose uptake, and LPL activity.