The development of an in vitro bioassay of high specificity, sensitivity and precision for the measurement of low circulating concentrations of biologically active glycoprotein hormones has offered exciting new insights into the in vivo secretion and metabolic clearance of luteinizing hormone (LH) in various pathophysiological states. Moreover, the most recent combined application of the rat interstitial cell testosterone (RICT) bioassay and a novel multiple-parameter deonvolution model has allowed investigators to dissect plasma concentration profiles of bioactive LH into defined secretory bursts, which have numerically explicit amplitudes, locations in time, and durations, and are acted upon by determinable subject- and study-specific endogenous metabolic clearance rates. Here, we have: (i) reviewed the ability of the endogenous GnRH pulse signal to regulate the in vivo secretion of biologically active LH molecules as assessed in the RICT and by deconvolution mechanics; (ii) demonstrated that low-dose exogenous GnRH pulses effectively mimic spontaneous bioactive LH pulsatility; (iii) investigated the role of endogenous androgen and estrogen in modulating bioactive gonadotropin secretion in men and women; and (iv) described significant alterations in endogenous LH bioactivity in puberty and healthy aging.