Burkholderia pseudomallei is the causative agent of melioidosis, a life-threatening disease common in Southeast Asia and northern Australia. Melioidosis often presents with nonspecific symptoms and has a fatality rate of upwards of 70% when left untreated. The gold standard for diagnosis is culturing B. pseudomallei from patient samples. Bacterial culture, however, can take up to 7 days, and its sensitivity is poor, at roughly 60%. The successful administration of appropriate antibiotics is reliant on rapid and accurate diagnosis. Hence, there is a genuine need for new diagnostics for this deadly pathogen. The Active Melioidosis Detect (AMD) lateral flow immunoassay (LFI) detects the capsular polysaccharide (CPS) of B. pseudomallei. The assay is designed for use on various clinical samples, including serum and urine; however, there are limited data to support which clinical matrices are the best candidates for detecting CPS. In this study, concentrations of CPS in paired serum and urine samples from melioidosis patients were determined using a quantitative antigen capture enzyme-linked immunosorbent assay. In parallel, samples were tested with the AMD LFI, and the results of the two immunoassays were compared. Additionally, centrifugal concentration was performed on a subset of urine samples to determine if this method may improve detection when CPS levels are initially low or undetectable. The results indicate that while CPS levels varied within the two matrices, there tended to be higher concentrations in urine. The AMD LFI detected CPS in 40.5% of urine samples, compared to 6.5% of serum samples, suggesting that urine is a preferable matrix for point-of-care diagnostic assays. IMPORTANCE Melioidosis is very challenging to diagnose. There is a clear need for a point-of-care assay for the detection of B. pseudomallei antigen directly from patient samples. The Active Melioidosis Detect lateral flow immunoassay detects the capsular polysaccharide (CPS) of B. pseudomallei and is designed for use on various clinical samples, including serum and urine. However, there are limited data regarding which clinical matrix is preferable for the detection of CPS. This study addresses this question by examining quantitative CPS levels in paired serum and urine samples and relating them to clinical parameters. Additionally, centrifugal concentration was performed on a subset of urine samples to determine whether this might enable the detection of CPS in samples in which it was initially present at low or undetectable levels. These results provide valuable insights into the detection of CPS in patients with melioidosis and suggest potential ways forward in the diagnosis and treatment of this challenging disease.