Early detection of disease and subsequent clinical intervention are central strategies in modern health care. Ongoing synergistic advances in medical understanding and bioanalytic technology are enabling scientists and clinicians to identify and quantify an increasing number of molecular biomarkers that reflect disease status and treatment effectiveness. Since enzymes play a key role in cellular functions such as signaling and metabolism, they are especially important as biomarkers of disease and indicators of likely prognosis. Low abundance and limited stability make it technically challenging to reliably detect enzymes in living subjects or in clinical samples such as excised tissue or biofluids such as urine or plasma. In addition, there is an emerging effort to develop enzyme assays that can be employed at the point of care where the technology must be affordable, sensitive, user-friendly, and deliverable. In this regard, optical-based methods are very attractive and there are many different assays to measure enzyme levels using colorimetric, UV-vis, fluorometric, or chemiluminescence detection techniques. However, these assays often suffer from technical drawbacks such as insensitivity, high cost, long preparation time, and poor selectivity. This thesis describes work that aims to eventually overcome these common limitations. Chapter 1 gives an overall introduction to the subject area by describing a range of methods for optical imaging and disease diagnosis, with a specific focus on N-acetyl-β-D-glucosaminidase (NAG) as an enzyme biomarker for various diseases including acute kidney injury disease (AKI), and the enzymes nitroreductase (NTR) and carbonic anhydrase IX (CA-IX) as biomarkers of tumor hypoxia. Chapters 2- 4 describe experiments that demonstrate new bioanalytical assays for early disease detection. Chapter 2 focuses on the optimization of a colorimetric assay that uses a supramolecular method to enhance colorimetric detection of urinary NAG. Chapter 3 describes a more user-friendly and accessible way to directly measure NAG levels and distinguish between healthy individuals versus patients with AKI . The method in Chapter 4 incorporates surface-enhanced Raman scattering (SERS) as a new and highly sensitive means of detecting glucosidase enzyme levels. Finally, Chapters 5, 6, 7 and 8 describes optical imaging and quantification of tumor hypoxia biomarkers using novel fluorescent near infrared molecular probes. The probes are highly specific and sensitive substrates for the clinically relevant hypoxia-associated enzymes, NTR and CA-IX.