The gonadotropin action mediated via three glycoprotein hormones, luteinizing hormone (LH), chorionic gonadotropin (CG) and follicle-stimulating hormone (FSH) is paramount for sexual differentiation, pubertal development and reproductive functions. The gonadotropins bind to their respective receptors, luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR), both belonging to G protein-coupled receptor (GPCR) family. The stimulation of gonadotropin receptors with their respective ligand leads, among other signaling pathways, to the production of cyclic 3’,5’ adenosine monophosphate (cAMP) through GαS-mediated adenylyl cyclase activation. Modelling of patient mutations or those created using site-directed mutagenesis has been instrumental in understanding structural-functional relationship of key residues affecting gonadotropin action. The current thesis characterizes a novel inactivating mutation in LH beta subunit of a patient as well as the development of two methods that were utilized for its molecular characterization. The first method, REPLACR-mutagenesis (Recombineering of Ends of linearized PLAsmids after PCR), is a one-step site-directed mutagenesis method, that utilizes in vivo recombineering for mutagenesis (deletions, additions and substitutions) in plasmid vectors. REPLACR-mutagenesis is an inexpensive alternative to commercial kits involving fewer steps and with similar efficiency. The second method, named CANDLES (Cyclic AMP iNdirect Detection by Light Emission from Sensor cells), was developed to monitor the kinetics of cAMP generation in cell cultures, without transfection of any real-time cAMP sensor, which is especially difficult in primary cell cultures. Finally, the LH beta mutation that causes a lysine (Lys20) deletion resulting in a hypogonadal phenotype in the patient was molecularly characterized with the above-mentioned methods.