During the last decade there has been an increased focus on the application of steroids as biomarkers in biomedical practice. The analysis of steroid hormones in biological samples of plasma or serum is currently routinely used in clinical diagnosis being an essential source of information on not only metabolic pathways, but also disorders of the metabolism. More importantly, the steroid hormones may reduce cancer development on the endocrinal basis, reveal abuse of anabolic substances, or even depression incidences. In the case of biomedical research, quantitative determination of steroids in serum or plasma creates an opportunity to diagnose diseases efficiently in their early stages and to monitor a patient for a possible recurrence of a disease after therapy. Therefore, an accurate measurement of steroids in the plasma or serum has become important for the contemporary medicine, even if troublesome especially in view of their low concentration in biological samples. The most common methods of steroid quantification in clinical practice nowadays include immunoassays such as radioimmunoassay or enzyme immunoassay. However, the main disadvantage of the immunoassay techniques consists in cross reactivity of the antibodies used in the assay with the related hormones. On the other hand, there is a number of applications where the analysis is performed with the use of modern separation techniques. The aim of this review is to present the development and applicability of the various analytical methods, all based on separation techniques and used for determination of steroids in the clinical laboratory, taking into account the recent progress in those areas. The review shows the advantages of high-performance liquid chromatography applied to determine non-volatile and thermally labile steroids and steroid conjugates. It also demonstrates that the currently used mass spectrometry offers practically useful structural information on the composition of steroids. A number of techniques and methods are compared and critically discussed pondering finally their specific analytical requirements like sensitivity, specificity, simplicity, limit of detection, and quantification. Nonetheless, plasma or serum sampling seems to remain one of the most convenient methods for population screening purposes.