Background: Feline interstitial cystitis (FIC) results from complex interactions between the urinary bladder, nervous system, adrenal glands and the cat's living environment. Following FIC, urolithiasis is the second most common cause of feline lower urinary tract disease (FLUTD), often producing clinical signs that mimic those of obstructive FIC. Objectives: This study aimed to assess the diagnostic utility of serum and urine concentrations of nerve growth factor (NGF) and glycosaminoglycans (GAGs) in cats with urolithiasis or interstitial cystitis, under both obstructive and non-obstructive conditions. Methods: The study included 90 cats, comprising 80 cats with clinical signs of FLUTD and 10 healthy controls. Group classification was based on clinical, laboratory and ultrasonographic examinations, as well as multimodal environmental modification (MEMO) evaluation criteria. Cats diagnosed with urolithiasis or FIC were further categorised into obstructive (urolithiasis/obstructive [URO/Ob], FIC/Ob) and non-obstructive (urolithiasis/non-obstructive [URO/Non-Ob], FIC/Non-Ob) subgroups. Results: Mild changes were noted in CBC and serum biochemistry. Serum and urine NGF levels were elevated in URO/Ob, FIC/Ob and FIC/Non-Ob groups but not in URO/Non-Ob. Serum and urine GAG levels were reduced in all groups except URO/Ob. In obstructive cases, serum (AUC = 0.693) and urine GAG (AUC = 0.629) showed moderate/acceptable diagnostic performance in distinguishing URO/Ob from FIC/Ob. In non-obstructive cases, serum (AUC = 0.622) and urine NGF (AUC = 0.612) demonstrated limited to moderate ability to differentiate FIC/Non-Ob from URO/Non-Ob. Conclusions: Elevated serum and urine NGF concentrations in cats with FIC have been associated with increased severity of bladder inflammation and tissue injury. In contrast, reduced urine GAG concentrations, observed in all groups except the URO/Ob group, may facilitate bladder inflammation by increasing urothelial permeability. Accordingly, serum and urine GAG levels in obstructive cases and serum and urine NGF levels in non-obstructive cases may provide clinically relevant insights into both the pathophysiology and diagnostic evaluation of FLUTD.