[en] The preparation of standards is possibly the single most important step in ensuring the clinical efficiency of an immunoassay. Therefore, there is a need for development of simple and efficient analytical methods to obtain analyte-free standards that resemble the original matrix. In particular, the determination of progesterone in milk and serum requires a standard completely free of the steroid, which becomes a very difficult task using conventional methods such as addition of charcoal or the use of special bovine milk. In this work, we report a procedure for the preparation of a progesterone- free standard using cloud point extraction and skim milk. The method involves the use of non-ionic surfactant (Triton X-114) as the extracting phase, and short centrifugation times. Different factors that can influence the extraction efficiency were evaluated using a radioimmunoassay (RIA) of progesterone in bovine milk. The RIA kit includes the use of a ¹²⁵I-labelled tracer purified by HPLC, a standard prepared with skim milk, and an anti-progesterone antiserum in solid phase. The method presented here can be used as a simple and low-cost alternative to obtain a standard containing progesterone at concentration levels below the detection limits of the immunoassay used.(Author)