Background <p>Noninvasive diagnostic biomarkers should be researched to reduce the need for imaging and radiation exposure in patients with kidney stone disease. This pilot study aimed to evaluate the diagnostic potential of urinary p-ERK/t-ERK (inflammatory signaling), osteopontin, CD44, and annexin II (adhesion molecule) levels in predicting kidney stone disease.</p> Methods <p>Thirty-seven participants were enrolled: 20 with and 17 without the disease. Spot urine samples were collected for crystal count and biochemical analyses. Enzyme-linked immunosorbent assay was performed to measure p-ERK/t-ERK in urine sediment and urinary osteopontin, CD44, and annexin II levels. Pearson’s correlation and receiver operating characteristic curve analyses were used to assess the correlations and diagnostic values of these biomarkers.</p> Results <p>The stone group showed significantly higher levels of p-ERK1/2 activity and urinary adhesion proteins (osteopontin, CD44, and annexin II) compared with those in the non-stone group. Receiver operating characteristic curve analysis revealed that these biomarkers could moderately distinguish between stone and non-stone groups. A multivariable logistic model combining CD44², ERK/CD44, and CD44/annexin II demonstrated enhanced discriminatory potential, achieving an AUC-ROC of 0.88. Additionally, annexin II levels showed a weak positive correlation with stone number and could distinguish patients with three or more stones from those with fewer.</p> Conclusions <p>This is the first study to reveal the concurrent elevation of ERK-related inflammatory signaling and urinary adhesion molecules in association with kidney stone disease. These markers could serve as potential candidate biomarkers for non-invasive diagnosis and disease monitoring. Our findings offer preliminary insights into the pathophysiology of kidney stones and may aid in future diagnostic development.</p>