Extraction is usually the first step to quantifying hormone levels in biological samples (blood, tissue, feces, yolk, saliva, water, etc.). In most cases, extraction is required to: (1) purify the sample and remove particulates that may interfere with your hormone assay; and (2) isolate and concentrate the hormone(s) of interest (particularly useful in low volume or low mass samples).
Many different extraction techniques have been developed for steroid and protein assays. The solvent used for extraction is chosen based on the analyte (hormone) you want to measure and your sample matrix (e.g. blood, feces, etc.). Extraction usually involves shaking and/or heating your sample to release the hormones from your sample matrix and into your solvent.
Solid phase extraction (SPE), which involves sending your sample down a sorbent-packed column, can be used to isolate hormones from water samples and other matrices. More information about SPE can be found here: Sigma Aldrich Guide to SPE
If you are interested in measuring multiple steroid hormones from a small sample, column chromatography can be used as part of your extraction method to isolate each analyte in question. Column chromatography consists of a stationary phase (a solid sorbent, such as celite or silica, packed in the column) and a mobile phase (a solvent mixture, which is run through the column). By loading your sample on the column and then adjusting the concentrations in your solvent mixture, you can separates out individual analytes based on their polarity. This results in individual fractions for each analyte of interest.
There are some assays that do not require extraction. These assays have been optimized to allow accurate measurement of hormone levels without interference from the sample’s matrix (e.g. components other than the hormone itself).