Copper (Cu) isotope ratios (⁶⁵Cu/⁶³Cu) in human biofluids have emerged as potential biomarkers for disease conditions associated with impaired Cu metabolism. Multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS) provides the precision required to detect and quantify the sub-permille differences in Cu isotope ratios between different biofluidic samples, but typically requires intensive sample pre-treatment to avoid isobaric interferences originating from the complex sample matrix (e.g., (⁴⁰Ar²³Na)⁺ on ⁶³Cu⁺, (⁴⁰Ar²⁵Mg)⁺ on ⁶⁵Cu⁺), all potentially inducing a bias of the true ratio. This contribution presents the development of a passive sampling method for Cu in human biofluids using the diffusive gradients in thin films (DGT) technique followed by ⁶⁵Cu/⁶³Cu isotope ratio analysis by MC-ICP-MS. The method is based on a Cu-selective polytetrafluoroethylene (PTFE) binding layer (CU membrane, TrisKem) designed for Cu isolation, aiming at simultaneous Cu preconcentration and matrix separation already in the sampling step. Eccrine sweat was used as an exemplary human biofluid with promising application potential for non-invasive medical diagnostics, potentially overcoming the drawbacks of invasive blood sampling such as patient discomfort and risk of infection. Deployment of the CU membrane in synthetic sweat spiked with Cu and other trace elements (Fe, Ni, Zn, Cd, and Pb) at physiologically relevant concentrations showed effective Cu uptake (96 % ± 2 %, n = 6) and separation from all other elements, particularly Na. The Na/Cu ratio was reduced from >4000 in the synthetic sweat to <0.1 in the DGT eluate, effectively mitigating the (⁴⁰Ar²³Na)⁺ isobaric interference on ⁶³Cu⁺. The methodological figures of merit including Cu recovery, selectivity, preconcentration, detection limits, and potential isotopic fractionation processes during DGT-based sampling and MC-ICP-MS measurement will be discussed in this presentation.