This work explores the use of readily available spectrophotometry to detect and monitor cyanobacteria and hexavalent chromium (Cr (VI)) under different water matrices. Research has shown that these parameters have been increasing in frequency worldwide with growing anthropological activities and aggravating climate change, resulting in unwanted source water contamination. The aim was to evaluate the potential of derivative spectrophotometry with changing cuvette pathlength (10-, 50-, and 100-mm) to be used as an early warning system for sensitive determination of the aforementioned parameters. Initially, Microcystis aeruginosa (cyanobacteria) and Chlorella vulgaris (green algae) were inoculated in deionized (D.I.) water individually and mixed in an equal concentration setting, respectively. The effect of increasing cuvette pathlength was investigated and results indicated a 15-, and 13-fold improvement in sensitivity with absorbance and derivative of absorbance from 10 mm to 100 mm pathlength, respectively. The lowest method detection limit (MDL) was observed using 100 mm and the concentration was found to be 4,916 cells/mL (for cyanobacteria), which is well below the WHO guideline for low probability of adverse health effects (< 20,000 cells/mL). Additionally, the mixed culture test demonstrated the potentiality of spectrophotometry to be able to identify cyanobacteria in mixed setting indicating applicability. Further microalgal testing was performed using longer pathlengths (50- and 100-mm), to investigate robustness of the developed methodology in surface water and under varying water quality parameters (WQPs) aka salinity, dissolved organic carbon (DOC), and turbidity, for realistic determination. 100 mm pathlength while employing derivative spectrophotometry were found to be most sensitive and concentration as low as 8,546 cells/mL could be detected. As expected, with increasing concentration of WQPs the sensitivity decreased, but overall, spectrophotometry was able to detect cyanobacteria in different water matrices. Lastly, similar methodology as before was exercised but it was applied for early detection of Cr (VI). Derivative spectrophotometry with longest pathlength was primarily utilized to investigate Cr (VI) response in D.I. water, pH water matrices, varying DOC, surface and tap water. Results indicated excellent MDLs as low as 2-, and 5-µg/L for tap and surface water, respectively, implicating practical viability.