AbstractWhen hospitalized, infants, particularly preterm, are often subjected to multiple painful needle procedures to collect sufficient blood for metabolic screening or diagnostic purposes using standard clinical tests. For example, at least 100 µL of whole blood is required to perform one creatinine plasma measurement with enzymatic colorimetric assays. As capillary electrophoresis–mass spectrometry (CE–MS) utilizing a sheathless porous tip interface only requires limited amounts of sample for in‐depth metabolic profiling studies, the aim of this work was to assess the utility of this method for the determination of creatinine in low amounts of plasma using residual blood samples from adults and infants. By using a starting amount of 5 µL of plasma and an injection volume of only 6.7 nL, a detection limit (S/N = 3) of 30 nM could be obtained for creatinine, and intra‐ and interday precisions (for peak area ratios) were below 3.2%. To shorten the electrophoretic separation time, a multi‐segment injection (MSI) strategy was employed to analyze up to seven samples in one electrophoretic run. The findings obtained by CE–MS for creatinine in pretreated plasma were compared with the values acquired by an enzymatic colorimetric assay typically used in clinical laboratories for this purpose. The comparison revealed that CE–MS could be used in a reliable way for the determination of creatinine in residual plasma samples from infants and adults. Nevertheless, to underscore the clinical efficacy of this method, a subsequent investigation employing an expanded pool of plasma samples is imperative. This will not only enhance the method's diagnostic utility but also contribute to minimizing both the amount and frequency of blood collection required for diagnostic purposes.