Evidence suggests menstrual cycle variation in the hormone relaxin may have an impact on ligament integrity and may be associated with risk of anterior cruciate ligament injury in physically active women. However, studies to date have only detected relaxin in a small number of participants, possibly due to inter-individual variability, frequency of sample collection, or analytical techniques. Therefore, the purpose of this study was to analyze serial serum samples in moderately active, eumenorrheic women to identify the proportion of women with detectable relaxin concentrations. Secondary analyses were conducted on two independent data sets. Data Set I (DSI; N = 66) participants provided samples for 6 days of menses and 8–10 days of the luteal phase. Data Set II (DSII; N = 15) participants provided samples every 2–3 days for a full menstrual cycle. Samples were analyzed via a relaxin-2 specific ELISA assay. Limit of detection (LOD) was calculated from the empirical assay data. LOD was calculated as 3.57 pg·ml−1. Relaxin concentrations exceeded the LOD in 90.91% (DSI) and 93.33% (DSII) of participants on at least 1 day of sampling. Actual peak values ranged from 0.0 pg·ml−1 to 118.0 pg·ml−1. Relaxin was detectable in a higher proportion of young women representing a broad range of physical activity levels when sampled more frequently. Future studies investigating relaxin should consider sampling on more than 1 day to accurately capture values among normal menstruating women.