Leaf biochemical and structural traits are vegetation characteristics related to various physiological processes. Taking advantage of the physical relationship between optical properties and leaf biochemistry, field-based spectroscopy has allowed for the rapid estimation of leaf biochemical constituents and repeated non-destructive measurements through time. Leaf constituent retrieval from leaf optical properties following inversion of the physically-based radiative transfer model PROSPECT is now a popular method, but some cases prompt poor retrieval success and this approach requires a strict inversion procedure. We investigated the performances of different inversion procedures for the estimation of leaf constituents, specifically chlorophyll a and b, carotenoids, water (EWT), and dry matter (LMA) from >1400 broadleaf samples, including the definition of optimal spectral subdomains, and the use of leaf reflectance or transmittance alone. We also developed a strategy to obtain prior information on the leaf structure parameter (N) in PROSPECT, when only reflectance or transmittance is measured, and examined the influence of this prior information in combination with different inversion procedures. We found that using the full domain of reflectance or transmittance only systematically leads to suboptimal estimation of chlorophyll a and b, carotenoids, EWT, and LMA, due to either the combined absorption of multiple constituents or inaccurate estimation of the N parameter. Our study confirms that the selection of optimal spectral subdomains leads to improved estimation of all leaf constituents, from 700 to 720 nm for chlorophyll a and b, 520–560 nm for carotenoids, and from 1700 to 2400 nm for EWT and LMA. Prior information on N, computed directly from the spectra, leads to systematic improved estimation of leaf constituents when only reflectance or transmittance is measured, with reductions in normalized root mean square error from 8 to 37%. We strongly recommend using optimal subdomains when inverting PROSPECT to retrieve leaf constituents, and with the availability of only reflectance or transmittance we further recommend the use of prior information on the N parameter.