Estrus synchronization is necessary for management of gilt reproduction in pig farms. It is usually achieved by using synthetic progestagens, but there is increasing demand for non-hormonal alternative tools with the prospect of sustainability of livestock production. Moreover, in organic farms, synthetic hormones are not allowed. Before reaching puberty, gilts exhibit a “waiting period” during which external stimulations, such as boar exposure, could trigger and synchronize the first ovulation. However, practical non-invasive tools for detection of the “waiting period” in pig farms are lacking. During this period, estrone levels in urine are high, but urine sampling is difficult in group-housed females. Our objective was to identify among steroids potential biomarkers of this “waiting period” through saliva monitoring from immature to pubertal gilts using gas chromatography coupled to tandem mass spectrometry. Starting between 144 and 147 days of age, six Large White gilts were submitted to ultrasound puberty diagnosis 3 times a week until first ovulation. Urine and saliva samples were collected to analyze weekly estrone and steroidome respectively, until puberty. Urinary estrone concentration significantly increased 2 weeks before first ovulation occurring between 182 and 192 days of age. The period with increasing estrone levels was considered as the “waiting period”. Steroidome analysis allowed identifying and quantifying 28 steroids in 500 µl of gilts saliva. Significant decrease of dehydroepiandrosterone and significant increase of 5α-dihydroprogesterone and 17β-estradiol were detected 2 weeks before puberty, suggesting that these steroids could be potential biomarkers of the “waiting period”. These results show that painless sampling of saliva could be a non-invasive welfare-friendly tool for the identification of the physiological hormonal status of the gilts and possibly the optimal time for application of the boar effect, a solution to synchronize puberty without exogenous hormones.