There is a need for rapid and sensitive non-sputum diagnostic tools for pulmonary and extrapulmonary tuberculosis (TB) in all patient populations. Lipoarabinomannan (LAM), a mycobacterial glycolipid, is a validated TB biomarker in urine, particularly in people living with HIV. However, LAM concentrations in patient samples are typically very low and existing assays have limited sensitivity. Quantitative, ultrasensitive detection of LAM may enable a novel approach for diagnosis and treatment monitoring. Methods: An ultrasensitive LAM assay was developed using Fluxus’ single-molecule counting platform, which integrates optics and microfluidics for biomarker detection. Limits of detection (LoD) and quantification (LoQ), dilution linearity, intra- and interassay precision, spike recovery, and matrix compatibility were determined. Clinical sample testing was performed using biobanked specimens from ten adults with suspected TB; patient sputum samples had been tested for presence of M. tuberculosis using smear microscopy, culture, and nucleic acid amplification test, and urine LAM concentration had been quantified using electrochemiluminescence assay. Testing in larger cohorts is ongoing.Results: LoD and LoQ were 0.068 and ~2 pg/mL, respectively, with a >5-log dynamic range. Mean spike recovery across 0.01–100 ng/mL was 106%, and dilution linearity was 102%. Intraassay and interassay CVs averaged 7.4% and <15%, respectively. No crossreactivity was observed with Mycobacterium smegmatis LAM. LAM was detected in urine from all patients with microbiologically confirmed TB (n=4) and not detected in non-TB patients (n=6), and concentrations were comparable to those observed using electrochemiluminescence.Conclusions: Fluxus’ ultrasensitive LAM assay demonstrates strong analytical performance and sub-pg/mL sensitivity. The assay detected LAM in urine from all patients with TB and was negative in all non-TB patients, and concentrations were comparable to those observed with a quantitative method. Fluxus’ platform supports the development of a non-sputum tool for TB diagnosis and treatment monitoring, addressing a critical gap in high-burden, resource-limited settings.