Rapid detection of expanded-spectrum cephalosporins (ESC) hydrolysing enzymes is crucial to implement infection control measures and antibiotic stewardship. Here, we have evaluated three biochemical ESC hydrolysis assays (ESBL NDP test, β-LACTA™ test, LFIA-CTX assay) and the NG-Test® CTX-M MULTI that detects CTX-M enzymes, on 93 well-characterized Gram-negative isolates, including 60 Enterobacterales, 21 Pseudomonas spp. and 12 Acinetobacter spp. The performances were good for all three hydrolysis assays, with the LFIA-CTX being slightly more sensitive and specific on the tested panel of isolates especially with Enterobacterales, without ambiguous results. This study showed that LFIA-CTX may be used for the detection of ESC hydrolysis as a competitive alternative to already available assays (β-LACTA™ test and ESBL NDP test) without any specific equipment and reduced hands-on-time. The lateral flow immunoassay NG-Test® CTX-M MULTI has proven to be a useful, easy, rapid, and reliable confirmatory test in Enterobacterales for detection of CTX-M-type ESBLs, which account for most of the resistance mechanisms leading to ESC resistance in Enterobacterales, but it misses rare ESC hydrolysing β-lactamases (AmpC, minor ESBLs, and carbapenemases). Combining it with the LFIA-CTX assay would yield an assay detecting the most frequently-encountered ESBLs (CTX-M-like β-lactamases) together with ESC hydrolysis.