A recent methodology, developed by our group, has enabled a dramatic improvement in the emissive nature of the excited species, formed during the chemiexcitation of dioxetanes under physiological conditions. This approach has resulted in the discovery of distinct phenoxy-dioxetane luminophores that produce a chemiluminescence signal via a direct-mode of emission. Here, we show a significant pKa effect of our new phenoxy-dioxetanes on their chemiexcitation and on their ability to serve as chemiluminescent turn-ON probes for biological applications. Using an appropriate phenoxy-dioxetane probe with a direct-mode of emission, we were able to image β-galactosidase activity, in cancer cells and in tumor-bearing mice. To the best of our knowledge, this is the first example to demonstrate in vitro and in vivo endogenous enzymatic chemiluminescence images obtained by a single-component phenoxy-dioxetane probe. We anticipate that our strategy, for the design and synthesis of such distinct luminophores, will assist in providing new effective turn-ON probes for non-invasive intravital chemiluminescence imaging techniques.