The impact of testosterone administration on the circulating lipidome in females remains unexplored, despite its relevance to understanding metabolic disorders like polycystic ovary syndrome (PCOS). This study addresses this gap by examining the effects of testosterone gel on the plasma lipidome of healthy women over three menstrual cycles. A cohort of 14 women aged 22-37 years with regular cycles was analyzed, with plasma samples collected at baseline, during peak testosterone levels (D45), and post-treatment (D59, D80). Testosterone gel treatment lasted 28 days, administered between day 29 and day 57 of the study. Using a deep-targeted lipidomic approach, 597 lipids were quantified to provide a detailed profile of the lipidome and capture subtle changes in lipid species and their associations with testosterone fluctuations. Extensive profiling revealed a significant decrease in 17 lipid species, especially ether- and ester-linked lysophosphatidylcholines (LPC), at peak testosterone. These lipid reductions were strongly negatively correlated with free and total testosterone, as well as dihydrotestosterone (DHT), and positively correlated with SHBG levels. Notably, intra-individual lipid variability was consistently lower than inter-individual variability, indicating a highly personalized lipidome regulation. Despite testosterone-induced changes, overall plasma lipidome alterations were minimal, suggesting mechanisms that maintain lipid homeostasis. This study highlights the complex interplay between testosterone and lipid metabolism in women. The minimal overall lipidome changes and high inter-individual variability point to the need for further research to assess the clinical relevance of these findings, particularly in hyperandrogenic conditions like PCOS. Clinical Trial Registration number: This study was registered on https://www.isrctn.com/ (ISRCTN10122130) on 09/01/2019.