Determining proline content accurately by spectrophotometry generally requires a standard calibration curve, so without it, quantification is unreliable; however, approximate estimation might be possible using literature-reported absorbance values and linear ranges, but this is not recommended for precise analysis.

It is generally not reliable to determine proline content using only spectrophotometric readings without a standard calibration curve, as the curve is critical for converting absorbance to concentration. Without a valid calibration, accurate quantification is challenging, and estimates based on literature slope values carry significant uncertainty.