Skeletal muscle toxicity has often been a critical concern in drug development, particularly with statins, which can cause adverse effects ranging from mild myalgia to fatal rhabdomyolysis. Traditional plasma biomarkers such as creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) have limitations in specificity and sensitivity. This study investigates urinary N-terminal titin fragment (N-titin) as a non-invasive biomarker for simvastatin-induced skeletal muscle toxicity in rats. Female Wistar Hannover rats were administered simvastatin at varying doses, and urine, blood, and muscle samples were collected for analysis. Histopathological examination revealed muscle fiber degeneration and inflammation in the fast-twitch muscles (gastrocnemius and biceps femoris) in high-dose rats. Correspondingly, urinary N-titin/creatinine (CRN) levels showed a marked increase, while plasma CPK and LDH levels remained unchanged. These findings suggest that urinary N-titin/CRN is a sensitive and practical biomarker for detecting skeletal muscle injury, potentially offering advantages over traditional blood-based markers.