Towards target diagnostics of low-concentrated molecules in biofluids, NMR spectroscopy faces limitations due to low sensitivity, signal overlap, and high equipment costs. Hyperpolarization methods such as photo-chemically induced dynamic nuclear polarization (photo-CIDNP) can mitigate some of these challenges. In this study, we explore the potential of steady-state photo-CIDNP to quantify target molecules in urine samples. Matrix interference poses a significant challenge to quantitative measurements, and we thus establish two counteracting methods: spiking and biofluid dilution. Experiments conducted in both high (14.1 T) and low (1.9 T) magnetic fields demonstrate the effectiveness of photo-CIDNP-based quantification at micromolar levels for the analytes sumatriptan and paracetamol. We report limits of quantification (LOQs) in complex matrices down to 3.5 μM and average errors of less than 26% with the spiking method and less than 11% using biofluid dilution. This proof-of-concept study highlights the potential of NMR supported by photo-CIDNP as a target diagnostic tool for rapid drug quantification and clinical monitoring applications, especially with low-cost benchtop NMR devices.