Supported Liquid Extraction technology is often used in the preparation of endogenous steroid hormones prior to analysis on LC-MS/MS. The extraction efficiency depends on the interphase formed between the aqueous sample and the organic extractant. Using the appropriate type of extractant and method is crucial for creating optimal conditions for analysis. The aim of the study was to reduce the amount of chemicals and time spent on preparation by altering the volume and type of extractant. Aldosterone, Androstenedione, 17-hydroxyprogesterone and 11-deoxycortisol, with predetermined regression and exchange criteria in mind, showed no significant variation between the reference method and the test method with reduced extraction volume, which indicates that the study can be used as basis for further validation. The slightly more sensitive steroid hormone Estradiol showed impaired yield and chromatography as a result of method modifications. This indicates that further tests regarding the type and proportions of extractant should be performed appropriately. This indicates that further tests regarding the type and proportions of extractant could be relevant.