of other diseases [2]. Commonly performed tests for cases where there is a suspicion of bladder cancer include cystoscopy (the \"gold standard\"), cytology of urinary sediment, and imaging diagnostics. However, these diagnostic methods have significant drawbacks: cystoscopy is invasive and uncomfortable for the patient, whereas laboratory tests have low sensitivity and specificity for early stages of bladder cancer [1]. These limitations have fostered research on alternative, more effective, minimally invasive approaches for bladder cancer diagnosis. As a first step in the creation of new diagnostic tools, untargeted metabolomics/metabolomic profiling of biological fluids can be used to identify biomarkers of bladder cancer [4], opening new paths towards the creation of novel biomarker-based diagnostic tools. Human urine is a biological matrix that is easy to obtain in large volumes. In addition to its easy availability, urine is an important biological matrix due to the type of information it may hold in relation to the various metabolic processes occurring throughout the human body. Urine is produced by the kidneys, which filter unwanted substances from the body; as such, urine contains many unnecessary and harmful by-products of metabolism. The main components of urine are water, urea, creatinine, ammonia, inorganic salts, and other water-soluble substances [5]. Given that urine is largely free from interfering lipids or proteins, sample preparation for this matrix is significantly less time-consuming in comparison to other biological matrices. Hence, metabolomic studies using urine as a biological matrix are promising [5]. Metabolites present in the urine, which are the final products of processes occurring in healthy and cancerous cells, can be of particular importance in the diagnosis of bladder cancer. Differences observed between metabolomic profiles of BC patients compared to healthy persons may allow us to find potential BC indicators [6]. Even though urine is a relatively simple biological matrix, it still requires careful sample preparation considerations with respect to issues such as matrix effects and sample-to-sample variations. The ideal sample preparation method should be simple (with the smallest number of steps) and reproducible. Besides, it must ensure sample purification, recovery of a wide range of analytes, as well as inhibition of metabolism, which protects compounds from degradation [7]. The simplest and most common preparation method of urine samples prior to MS analysis is dilution (\"dilute and shoot\"). However, this simplistic strategy compromises sensitivity and does not ensure metabolism quenching, thus spurring the development of alternative approaches to urine sample preparation to address these shortcomings. Of the variety of methods developed for urine analysis, solid-phase microextraction has been demonstrated to largely fulfill the abovementioned conditions, given that in addition to integrating various steps of the sample preparation and extraction process, it additionally provides efficient sample clean-up, making it compatible with liquid chromatography and mass spectrometry [7; 8]. In addition, the technology offers flexibility in terms of device geometry, chemistry of the extraction phase, and high-throughput capabilities. With regards to the latter, the most common format of the device i.e., the microfiber, is widely used for low-throughput analysis of tissues or in animal studies [9; 10], as well as for high throughput analyses where sample volumes are limited [11]. When sample volume is of no concern, the thin film format of SPME (TFME) is preferable, as it increases recovery of analytes and is compatible with commercial automated or semi-automated high throughput robotic systems [10]. In the current study, thin film SPME in high throughput semi-automated mode was used for metabolomics screening of urine of patients with bladder cancer and healthy controls.