Steroids are synthesized in several tissues (e.g. adrenal glands, brain, skin) and regulate essential biological functions including growth and reproduction. Disruptions in steroid homeostasis can lead to serious physiological problems. Thus, protocols have been developed to measure steroids in biological matrices such as follicular liquid or periprostatic adipose tissue. It is a real challenge to be able to quantify them rapidly in various matrices. Today, GC-MS/MS analyses is the main technology used as it allows the quantification of an extended list of steroids (Androgens, Estrogens and Progestagens). These GC-MS/MS methods are very efficient in terms of sensitivity but remain long and expensive. Thus, a new method was studied and it was possible to create a faster and less expensive method that allows to enlarge the list of steroids available for analysis with the detection of molecules belonging to the corticoid family by HPLC-HRMS with online SPE system. These two techniques are discussed and evaluated in particular from the point of view of performance, practicality of sample preparation, molecules accessible to analysis.