<p>In biomedical research, it is essential to be able to precisely and reliably quantify trace and microelements in complex biological matrices in order to identify deficiencies and understand disease mechanisms. Analytical methods that are sensitive, robust, and environmentally friendly are preferable. Suspension-assisted total reflection X-ray fluorescence spectrometry (SA-TXRF) is a simple to perform, multielement analytical method that can quantify trace elements in suspensions containing minute sample amounts. In this work, a systematic study comparing five different suspension reagents, namely concentrated and diluted nitric acid, Triton X-100, Triton X-114, and ultrapure water, for SA-TXRF analysis of trace and microelements in three biological reference materials (NIST 1577c, NIST 1486, and ERM-BB186) is presented. The analytical performance is compared in terms of trueness, precision, and sensitivity, and the resulting SA-TXRF methods were assessed for greenness using the Analytical Greenness Metric for Sample Preparation (AGREEprep). As a result, water is recommended as suspension reagent for soft biological material, while for hard biological material, i.e., bone meal, best results were achieved using the surfactant Triton X-114. All of the presented SA-TXRF methods demonstrate improved greenness compared to the standard method (EPA Method 3052), which involves sample preparation by microwave-assisted digestion.</p> Graphical abstract <p></p>