Collecting urine samples in neonates by catheterization or suprapubic puncture cause trauma, while self-adhesive collection bags can damage fragile skin. An alternative method is the collection of samples from urine-soaked cotton wool balls placed in diapers.To compare the concentration of albumin, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), and uromodulin between clean catch urine and samples collected in cotton balls in neonates and assess the efficiency of exosome extraction.Standard clean catch urine samples (SS) were assayed for albumin, creatinine, NGAL, uromodulin by commercial ELISA kits. Concentrations were compared to the same urine samples extracted immediately from soaked cotton wool balls (sample 2, S2) or urine extracted from cotton wool balls placed in diaper in a warm incubator for 2 hours before extraction (sample 3, S3). Exosomes were extracted from all three samples of one patient for visualization under electron microscopy.26 infants (17 males) of median gestational age at birth of 32+1 weeks had urine collected at a median age of 29 days at 37+ 6 weeks corrected age. Concentrations in S2 and S3 were within 10% of concentration of SS in 46% and 35% of specimens for albumin, 69% and 58% for creatinine, 12% and 12% for NGAL, 27% and 15% for uromodulin, without consistent positive or negative bias. Urine albumin/creatinine ratios (UACR) were 4.3% less in S2 and 4.5% less in S3 compared to SS. Exosomes were extracted and visualised from all three sample types.Neonatal urine samples extracted from cotton wool balls can be used to screen for relevant albuminuria, but provide imprecise estimates of NGAL and uromodulin, The proof of exosome extraction from urine collection in cotton wool balls opens the potential to examine exosomal cargo.